Journal of Controlled Release 196 (2014) 243–251

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Nano-engineered mesenchymal stem cells as targeted therapeutic carriers Tanmoy Sadhukha a, Timothy D. O'Brien b, Swayam Prabha a,c,⁎ a b c

Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55455, USA Stem Cell Institute and Veterinary Population Medicine Department, University of Minnesota, 1365 Gortner Avenue, Saint Paul, MN 55108, USA Center for Translational Drug Delivery, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55455, USA

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Article history: Received 8 July 2014 Accepted 15 October 2014 Available online 23 October 2014 Keywords: Nanoparticles Mesenchymal stem cells Chemotherapy Targeted drug delivery Drug efflux

a b s t r a c t Poor availability in deep-seated solid tumors is a significant challenge that limits the effectiveness of currently used anticancer drugs. Approaches that can specifically enhance drug delivery to the tumor tissue can potentially improve therapeutic efficacy. In our current studies, we used nano-engineered mesenchymal stem cells (nanoengineered MSCs) as tumor-targeted therapeutic carriers. In addition to their exquisite tumor homing capabilities, MSCs overexpress efflux transporters such as P-glycoprotein and are highly drug resistant. The inherent tumor-tropic and drug-resistant properties make MSCs ideal carriers for toxic payload. Nano-engineered MSCs were prepared by treating human MSCs with drug-loaded polymeric nanoparticles. Incorporating nanoparticles in MSCs did not affect their viability, differentiation or migration potential. Nano-engineered MSCs induced dosedependent cytotoxicity in A549 lung adenocarcinoma cells and MA148 ovarian cancer cells in vitro. An orthotopic A549 lung tumor model was used to monitor the in vivo distribution of nanoengineered MSCs. Intravenous injection of nanoparticles resulted in non-specific biodistribution, with significant accumulation in the liver and spleen while nano-engineered MSCs demonstrated selective accumulation and retention in lung tumors. These studies demonstrate the feasibility of developing nano-engineered MSCs loaded with high concentration of anticancer agents without affecting their tumor-targeting or drug resistance properties. Published by Elsevier B.V.

1. Introduction Encapsulation in colloidal delivery systems has been investigated extensively as a means to improve the delivery of anticancer drugs in tumor tissues and to reduce the drug exposure in normal organs [1]. Such delivery systems attempt to exploit the enhanced permeation and retention (EPR) effect to passively accumulate in tumors [2]. However, there is increasing recognition that the EPR effect may not be a universal phenomenon and is likely restricted to tumors that are well perfused [3]. Even within highly vascularized tumors, the existence of under-perfused and hypoxic regions has been documented [4]. Such tumors are less likely to benefit from the EPR effect. Active targeting approaches utilize ligands that can bind with surface antigens or receptors that are overexpressed on tumor cells [5]. However, active targeting is, in reality, not an active process, since the delivery system must first accumulate passively in the tumor, followed by binding to tumor cells. Thus, active targeting suffers from some of the same limitations as

⁎ Corresponding author at: Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard St SE, WDH 9-151, Minneapolis, MN 55455, USA. Tel.: +1 612 626 3545; fax: +1 612 626 2125. E-mail address: [email protected] (S. Prabha).

http://dx.doi.org/10.1016/j.jconrel.2014.10.015 0168-3659/Published by Elsevier B.V.

passive targeting [6]. Thus, there is a clear need for delivery systems that can target tumors actively, independent of the EPR effect. Mesenchymal stem cells (MSCs) have been shown to actively traffic to tumors, in response to inflammatory signals secreted by neutrophils and macrophages invading the tumor [7]. Further, MSCs can be genetically modified to express peptides and proteins with anti-tumor properties [8]. For example, MSCs expressing interleukin (IL)-12 were shown to inhibit the growth of melanoma, breast and hepatoma tumors [9]. Thus, the tumortropic nature of MSCs enables the possibility of true, active tumor targeting of anticancer agents. However, their use as delivery vehicles for cytotoxic drugs has been limited by their overexpression of drug efflux transporters such as P-glycoprotein, which results in poor intracellular drug accumulation [10]. In addition, small molecules are also subject to rapid diffusional clearance from cells. We hypothesized that incorporation in the nanoparticleencapsulated form will enable successful loading and retention of cytotoxic drugs in MSCs. Drug-loaded nanoparticles are readily formulated with the FDA-approved, biodegradable and biocompatible polymer, poly (DL-lactide-co-glycolide) (PLGA) [11]. Previous studies have shown that PLGA nanoparticles are internalized into cells through endocytosis and a fraction of the internalized particles escape the endolysosomal pathway to reach the cytoplasm [12]. Nanoparticles that are retained within the cells act as intracellular drug depots, slowly

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releasing the encapsulated drug. Thus, MSCs loaded with drugcontaining nanoparticles (nano-engineered MSCs) should be capable of actively accumulating in tumors and slowly releasing the drug, resulting in effective inhibition of tumor growth. The overall objective of this study was to investigate PLGA-loaded nanoparticles for loading a cytotoxic drug, paclitaxel, in MSCs and to determine the ability of nano-engineered MSCs to target the tumor tissue in a mouse orthotopic lung tumor model. 2. Materials and methods 2.1. Materials Paclitaxel, polyvinyl alcohol (PVA), ammonium acetate, chloroform, beta-glycerophosphate, ascorbic acid, dexamethasone, 3-isobutyl-1methylxanthine, indomethacin, insulin, alizarin red and oil red O were purchased from Sigma (St. Louis, MO). Penicillin/streptomycin and fetal bovine serum were purchased from Bioexpress (Kaysville, UT). RPMI 1640, alpha-MEM, Dulbecco's phosphate buffered saline (DPBS), trypsin-EDTA solution and 7-aminoactinomycin D (7-AAD) were obtained from Invitrogen Corporation (Carlsbad, CA). Ester-terminated 50:50 poly(DL-lactide-co-glycolide) (inherent viscosity: 0.95–1.2 dl/g) was purchased from Lactel Absorbable Polymers (Birmingham, AL). Coumarin-6 was purchased from Acros Organics. Methanol, acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA). Captisol was acquired from Captisol® (La Jolla, CA). Mesenchymal stem cell media was purchased from ScienCell Research Laboratories (Carlsbad, CA). Near-infrared dye SDB 5491 was purchased from HW Sands (Jupiter, FL).

C18 column (Supelco 4.6 × 150 mm, 5 μm) in isocratic mode. The mobile phase consisted of 60% of 10 mM ammonium acetate (pH 4) and 40% acetonitrile at a flow rate of 1 ml/min. Paclitaxel eluted at a retention time of 4 min and was quantified by UV detection at 228 nm [14]. Paclitaxel solutions in methanol (100 ng/ml–10 μg/ml) were used as standards to construct a standard curve. Paclitaxel release from nanoparticles was assayed in cell culture medium supplemented with Captisol®. Nanoparticle dispersion (0.1 mg/ml) in RPMI containing 10% w/v Captisol® was kept at 37 °C and 150 rpm for 2 weeks. Aliquots of the dispersion (500 μl) were withdrawn at 1 h, 6 h, 1 day, 2 days, 5 days, 7 days, 9 days, 11 days and 14 days. The aliquots were centrifuged at 13,000 rpm for 15 min and 450 μl of the supernatant was collected and analyzed directly for paclitaxel content by HPLC. Paclitaxel solution in RPMI supplemented with 10% Captisol® maintained under the same conditions and duration was used as HPLC standards. 2.4. Cell culture studies MA148 (ovarian cancer) cells were received as a gift from Dr. Sundaram Ramakrishnan, A549 (human lung adenocarcinoma) cells were obtained from Dr. Jayanth Panyam and human-MSCs were obtained from ScienCell Research Laboratories (Carlsbad, CA). MA148 and A549 cell lines were propagated using RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin antibiotic solution. Human-MSCs were propagated using mesenchymal stem cell medium from ScienCell. All the cells were maintained at 37 °C and in 5% carbon dioxide. 2.5. Preparation of nano-engineered MSCs

2.2. Synthesis and characterization of PLGA nanoparticles Paclitaxel and a fluorescent probe (coumarin-6 for in vitro studies or SDB 5491 for in vivo imaging) were loaded in PLGA nanoparticles by emulsion-solvent evaporation technique [13]. In brief, 5 mg of paclitaxel and 100 μg of coumarin-6 (green fluorescent) or 50 μg of SDB 5491 (near-infrared fluorescence) were dissolved in 1 ml of chloroform containing 32 mg PLGA. This organic phase was emulsified 7.5 ml of 2.5% w/v polyvinyl alcohol and sonicated at 20 W power for 35 min to form the emulsion. Chloroform was evaporated by stirring overnight under ambient conditions, followed by one hour stirring under vacuum. The nanoparticle dispersion was washed three times by ultracentrifugation at 148,000 ×g for 35 min (Optima XPN-80 Ultracentrifuge, Beckman Coulter, Chaska, MN). After the final wash, nanoparticles were dispersed in purified water and centrifuged at 1000 ×g for 6 min. The supernatant was lyophilized (Labconco, FreeZone 4.5, Kansas City, MO). Control nanoparticles containing coumarin-6 but no paclitaxel were also prepared similarly. 2.3. Characterization of nanoparticles The hydrodynamic diameter of nanoparticles was determined using photon correlation spectroscopy. Nanoparticles dispersed in deionized water (~ 0.1 mg/ml) were subjected to particle size analysis using a Delsa™ Nano C Particle Analyzer (Beckman, Brea, CA). Measurements were performed at a 165° scattering angle at 25 °C. Five sets of size measurement runs recording 150 size events per run were performed for each sample. The mean hydrodynamic diameter was calculated based on size distribution by weight, assuming a lognormal size distribution. Zeta potential of nanoparticles was also determined using the same instrument. The results were expressed as mean ± S.E.M. of five runs. Paclitaxel loading in nanoparticles was analyzed by dispersing 1 mg of nanoparticles in 1 ml of methanol and extracting overnight using a rotary extractor. The methanolic extract was centrifuged at 13,000 rpm for 30 min to remove particle debris. Quantification of paclitaxel was performed by high-performance liquid chromatography (HPLC) using a

MSCs were trypsinized, suspended in cell culture media at 250,000 cells per ml and incubated with 1 ml of 100 μg/ml of nanoparticles for 4 h at 37 °C with occasional mixing. After 4 h, unencapsulated nanoparticles were removed by 3 washes with Dulbecco's phosphate buffered saline (DPBS) followed by centrifugation at 1000 rpm for 5 min. The final cell pellet was resuspended in growth medium and used for further studies. 2.6. Uptake and cytotoxicity of nanoparticles in MSCs Intracellular uptake of fluorescently tagged nanoparticles in MSCs was analyzed qualitatively by fluorescence microscopy and flow cytometry and quantitatively by analyzing the amount of nanoparticles in the MSCs by HPLC. For flow cytometric analysis, MSCs were suspended in cell culture media supplemented with 5% FBS at a density of 50,000 cells per ml. Nanoparticles were added at a concentration of 50 μg/ml and the mix was maintained at 37 °C. 7-Aminoactinomycin D (7-AAD) was added to the tubes as a cell viability marker. Flow cytometric analysis was performed at 30 min, 1 h, 2 h, 4 h and 5.5 h after nanoparticle addition. Nanoparticle-associated green fluorescence in the cells was detected in the FL-1 channel while 7-AAD fluorescence was detected in the FL-3 channel of the flow cytometer (BD, FACSCalibur™). Data from 10,000 cells in each group were analyzed using FlowJo™ software. Quantitative analysis of cellular uptake was performed in both suspended and plated cells. MSCs in suspension (~100,000 cells/ml) were incubated with 100 μg/ml of nanoparticles for 15 min, 1 h, 2 h, 4 h and 6 h at 37 °C. At each time point, the cell suspension was washed 3 times with 1 ml of DPBS to remove the non-internalized nanoparticles. The final cell pellet was resuspended in 125 μl of DPBS and lysed by 2 freeze thaw cycles. Cell debris was centrifuged at 17,000 ×g for 15 min. 25 μl of the supernatant was used for protein quantitation by bicinchoninic acid assay (Thermo Scientific). Coumarin-6 and paclitaxel were extracted from the cell lysate with 500 μl of methanol for 2 h. The methanolic extract was centrifuged at 17,000 ×g for 15 min and the

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supernatant was analyzed for paclitaxel content by HPLC and coumarin-6 content by fluorescence spectroscopy. Paclitaxel and coumarin-6 content were normalized to total cell protein to calculate nanoparticle uptake in cells. For quantitative analysis of plated MSCs (50,000/well), 500 μl of 100 μg/ml nanoparticles dispersed in cell culture media was added to the cells and incubated for 15 min, 1 h, 2 h, 4 h and 6 h at 37 °C. At the end of the incubation, the cells were washed 3 times with 1 ml DPBS, 200 μl of DPBS added to the wells, and the cells were lysed by 2 freeze thaw cycles. The cell lysate was collected and processed as described above. For microscopic analysis, cells were plated in a 24-well plate at 30,000 cells per ml. Cells were incubated with 50 μg/ml or 100 μg/ml of nanoparticles at 37 °C for 30 min, 1 h, 2 h, 4 h, 6 h or 24 h. At each time point, cells were washed three times with DPBS and imaged using a Zeiss Axioplan fluorescence microscope using a FITC filter. Toxicity of paclitaxel-loaded nanoparticles to MSCs was evaluated by incubating the MSCs (5000 cells/well) with 100 μg/ml of nanoparticles for 5 days. Relative survival of MSCs was determined on days 3 and 5 by using MTS cell viability assay. 2.7. Drug release from nano-engineered MSCs Nano-engineered MSCs, prepared as described above, were washed 3 times and then incubated in cell-culture media (50,000 cells in 500 μl) containing 1% captisol for 15 min, 1 h, 2 h, 4 h and 6 h and 24 h at 37 °C. At each time point, the cell suspension was centrifuged at 1000 rpm for 6 min to remove the cells and 450 μl of the supernatant was removed, extracted with 500 μl of acetonitrile and analyzed by HPLC for paclitaxel content. Drug release was plotted as a percent of the total drug loaded in 50,000 nano-engineered MSCs. 2.8. Efflux potential of MSCs P-glycoprotein (P-gp) activity in nano-engineered MSCs was evaluated using calcein AM as P-gp substrates. Nano-engineered MSCs were incubated with 0.25 μM calcein AM at 37 °C for 30 min in the presence or absence of 1 μM Zosuquidar (LY335979), a specific inhibitor of P-gp. Calcein concentration inside the cells was determined using a fluorescent plate reader (λex: 494 nm and λem: 517 nm). 2.9. Cytotoxicity study Cytotoxic potential of nano-engineered MSCs against A549 lung tumor cells was evaluated using a Transwell® assay. A549 cells were seeded at the bottom of Transwell® plates at 7500 cells per well in 100 μl. Nano-engineered MSCs were added to the top of the Transwell® insert at different cell densities. A549 cell viability was determined by MTS assay on days 3 and 5. To confirm the cytotoxic potential of nano-engineered MSCs, the study was repeated in MA148 ovarian tumor cells using the same method. Tumor cells that were treated with non-drug loaded MSCs or just cell culture medium were used as controls. Additionally paclitaxel solution and paclitaxel loaded nanoparticles were also used as positive controls. The cell viability data was fit to a sigmoidal dose response curve using GraphPad Prism software (GraphPad Software, Inc.) to determine the IC50 of the different treatments.

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dexamethasone. Adipogenic medium consisted of alpha-MEM supplemented with 10% FBS, 1% antibiotics, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 100 mM indomethacin and 10 mg/ml insulin. MSCs treated similarly but without the initial nanoparticle incubation step were used as untreated control. After 3 weeks, the presence of calcium deposits was detected by alizarin red staining in 70% ethanolfixed cells while neutral lipid vacuoles were detected by oil red O staining in 4% formalin-fixed cells to assess osteogenic and adipogenic differentiation potentials of stem cells, respectively. MSCs grown in regular growth medium were also stained similarly and used as negative control. Images of the cells were captured using an inverted light microscope (Zeiss Axiovert 40 CFL), at 100× magnification. 2.11. In vitro migration potential of nano-engineered MSCs Migratory potential of nano-engineered MSCs was compared to untreated MSCs using a Transwell® plate. MSCs were serum starved for 24 h prior to the study. Nano-engineered MSCs were generated as described previously in serum-free condition. Following DPBS washes, and resuspension in serum-free medium, 5000 untreated MSCs or nano-engineered MSCs were added to the top well of a 96-well Transwell® plate containing 8.0 μm pore size PET membrane (Corning Life Sciences). Culture media containing 5% serum or serum-free media were added to the bottom well. Cell migration was allowed to happen at 37 °C for 20 h. After the designated time, the top and bottom wells were washed with DPBS and 1.2 μg/ml calcein AM solution in cell dissociation media was added to the bottom well and incubated for 1 h at 37 °C with light tapping after 30 min to dislodge the migrated cells. The cell suspension was transferred to a black-walled 96-well plate and the fluorescence was read at 485 nm/520 nm. A standard curve of the untreated and nano-engineered MSCs treated with calcein AM was used to quantify the number of migrated cells. 2.12. Orthotopic lung tumor model The animal study was performed in compliance with a protocol approved by the Institutional Animal Care and Use Committee at the University of Minnesota. A mouse orthotopic lung tumor model was used in the studies. Female Fox Chase SCID® Beige mice (CB17.Cg-PrkdcscidLystbg-J/ Crl), four to five weeks of age, were obtained from Charles River Laboratories. A549-luc-C8 Bioware® Cell Line (Caliper Life Sciences) is a luciferase expressing cell line derived from A549 human lung carcinoma cells. Intravenous injection of 0.5 million A549-luc-C8 cells led to detectable increase in bioluminescence in the lungs by 2 weeks [16]. 2.13. In vivo distribution of nano-engineered MSCs Animals bearing A549 lung tumors were injected with 150 mg/kg of intraperitoneally and the resulting bioluminescence imaged using Xenogen IVIS Spectrum live animal imager, to confirm the presence of lung tumors. Untreated MSCs or nano-engineered MSCs were injected intravenously at 250,000 cells/100 μl/mice (n = 5 per group) and the resulting fluorescence profiles were imaged at 15 min, 1.5 h, 3 h and 24 h post injection. Tumor bearing animals injected with saline were also imaged as controls. The fluorescence images were captured and analyzed using Living Image® software.

D-luciferin

2.10. Differentiation potential of nano-engineered MSCs 3. Results Effect of nanoparticle uptake on adipogenic or osteogenic differentiation potential of MSCs was studied. MSCs plated in 6-well plates (250,000 cells/well) were incubated with 100 μg/ml nanoparticles for 4 h at 37 °C, washed with DPBS and incubated with adipogenic or osteogenic differentiation media [15] for 3 weeks. Osteogenic medium comprised of alpha-MEM supplemented with 10% FBS, 1% antibiotics, and 10 mM beta-glycerophosphate, 100 mM ascorbic acid, and 10 mM

3.1. Nanoparticle characterization PLGA nanoparticles containing paclitaxel and a fluorescent dye were formulated. The average hydrodynamic diameter of nanoparticles was 259 ± 2 nm and the polydispersity index was 0.093. The zeta potential of nanoparticles was −17.1 ± 2.1 mV. Paclitaxel was efficiently loaded

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in nanoparticles (148 ± 5 μg/mg; average encapsulation efficiency = 93.5 ± 3.5%). In vitro release of paclitaxel from nanoparticles is shown in Fig. 1. Following an initial burst release of about 25%, the release rate was steady at about 5% release per day. Greater than 90% of the encapsulated drug was released in 9 days.

3.2. Uptake and cytotoxicity of nanoparticles in MSCs Quantitative analysis of nanoparticle uptake in MSCs is shown in Fig. 2A. Nanoparticle uptake in MSCs was found to reach a steady state between 4 and 6 h. Further incubation for 24 h did not improve the cellular levels of nanoparticles (Fig. 2A). Four hours after incubation with 100 μg/ml of nanoparticle dispersion, the nanoparticle load in the MSCs was 327 ± 27 μg/mg cellular protein. For direct estimation of paclitaxel load in the MSCs, known number of live MSCs incubated with nanoparticles were extracted and analyzed for the amount of drug. The average paclitaxel content was 4.7 pg/cell. Flow cytometry was also used to monitor the uptake of fluorescently labeled nanoparticles in MSCs. Cellular uptake of nanoparticles increased in time-dependent manner (Fig. 2B). Time-dependent increase in intracellular fluorescence was also observed in nanoparticle-treated cells by fluorescence microscope (Fig. 2C–G). Nanoparticle uptake did not affect the viability of MSCs, as evidenced by the lack of 7-AAD uptake in nanoparticle-treated cells (Fig. 2B). MSCs treated with a nanoparticle dose of 100 μg/ml for 4 h were used in further studies. Paclitaxel release from nano-engineered MSCs was sustained, with approximately 2.1% of the encapsulated drug released in the first 24 h (Fig. 2N). Calcein assays were used to characterize the presence of efflux activity in MSCs. Cellular accumulation of calcein AM, a P-gp substrate, was significantly enhanced by Zosuquidar, a specific P-gp inhibitor (Fig. 2M). These studies confirm that nano-engineered MSCs retain Pgp-mediated efflux activity and suggest that paclitaxel can be released from nano-engineered MSCs through P-gp-mediated efflux. Long term effect of nanoparticle uptake on MSC survival was studied on adherent cells. Nanoparticle uptake did not significantly affect the survival of nano-engineered MSCs after 3 and 5 days (Fig. 3A). MSC survival at 3 and 5 days was unaffected after incubation with identical concentration of paclitaxel in solution (equivalent to that contained in 100 μg/ml nanoparticles) as well. This further confirmed the resistance of MSCs to paclitaxel and the suitability of nano-engineered MSCs as a delivery vehicle for cytotoxic drugs like paclitaxel. The small reduction in viability (b 20%) after 5 days could possibly be because of paclitaxel released within the MSCs overwhelming P-gp efflux.

3.3. Cytotoxicity studies The cytotoxic potential of nano-engineered MSCs was determined in MA148 and A549 cells. Different doses of nano-engineered MSCs were added on the top donor wells of a Transwell® plate, with the adherent tumor cells seeded in the bottom chamber. MTS analysis of the tumor cells at 3 days and 5 days after MSC treatment revealed dosedependent decrease in cell survival (Fig. 3B, C). IC50 values for A549 cells were 2025 ± 1 and 2448 ± 1 nano-engineered MSCs after 3 and 5 days, respectively, while that for MA148 cells, it was 1401 ± 1 and 1478 ± 1 cells, respectively (Fig. 3D). Untreated MSCs or MSCs loaded with blank nanoparticles did not affect the survival of tumor cells (Supplementary Fig. 1). IC50 of free paclitaxel and paclitaxel encapsulated in nanoparticles was 1.73 nM and 6.71 nM, respectively, in A549 cells and 0.96 nM and 4.52 nM, respectively, in MA148 cells (Fig. 3E). 3.4. Differentiation and migration potentials of nano-engineered MSCs Osteogenic and adipogenic differentiation potentials of nanoengineered MSCs were compared with untreated MSCs. To minimize the risk of paclitaxel-induced loss of cell viability, control nanoparticles without PTX was used in this study. Lipid filled vacuoles, characteristic of adipogenic differentiation, were seen in both untreated and nanoengineered MSCs grown in adipogenic differentiation medium for 3 weeks (Fig. 4A, C). Similarly, osteogenic differentiation, characterized by calcium deposits in the cells after 3 weeks of culturing in osteogenic differentiation medium, was seen in both untreated MSCs and nanoengineered MSCs (Fig. 4B, D). In contrast MSCs grown in regular growth media (control) showed no differentiation (Fig. 4E, F). This suggests that the differentiation properties of MSCs remain unaffected by nanoparticle treatment. To compare the migratory potential of nano-engineered MSCs with that of untreated MSCs, 5000 cells from each group were allowed to migrate towards FBS rich or serum-free media for 20 h. While minimal migration was observed in either group towards serum-free media (Fig. 4G), the migratory behavior of both the untreated and nanoengineered MSCs towards serum was similar with no statistical difference (Fig. 4G, Supplementary Fig. 2). About 18% of the cells migrated to the bottom compartment in 20 h in both treatment groups. 3.5. Animal studies In vivo biodistribution of nano-engineered MSCs was compared with that of nanoparticles in an orthotopic lung tumor model (Fig. 5A). Infrared fluorescence profile of nano-engineered MSCs was remarkably different from that of nanoparticles (Fig. 5B). Nano-engineered MSCs remained primarily in the lung for at least 3 h post intravenous injection. After 24 h, diffuse fluorescence was also detectable in the abdominal region (Supplementary Fig. 3). In contrast, 10 min after injection of nanoparticles, strong fluorescence was visible in various regions of the body including the lungs and abdomen. Within 1.5 h almost all fluorescence from the thoracic area had disappeared and instead strong fluorescence signal was detectable in the abdominal organs. Abdominal fluorescence was also visible after 24 h with some fluorescence in kidneys and bladder. 4. Discussion

Fig. 1. Paclitaxel release from nanoparticles. Paclitaxel nanoparticles were dispersed in cell culture medium supplemented with Captisol® and at set times, an aliquot of the dispersion was centrifuged and the supernatant analyzed by HPLC for paclitaxel content. Data shown is mean ± S.D., n = 3.

MSCs possess several unique characteristics, which make them attractive carriers for cancer therapy. MSCs have been shown to have low immunogenicity and a positive safety profile in clinical trials to date [17–19]. Their biodistribution has been studied extensively and their ability to selectively home to sites of inflammation and cancer has been previously documented [20,21]. Based on their tumor targeting capabilities, a number of studies have investigated genetically modified MSCs to deliver peptides and proteins to tumors [9]. However,

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Fig. 2. Cellular uptake of nanoparticles. (A) Quantitative analysis of the cellular uptake of nanoparticles. MSCs were incubated with 50 or 100 μg/ml of drug loaded nanoparticles. At different time points, the cells were washed, lysed and the cell lysate assayed for protein content and paclitaxel concentration. Data shown is mean ± S.D., n = 3. (B) Cellular uptake of nanoparticles and subsequent cell viability assayed by flow cytometry. MSCs suspended in media were incubated with 100 μg/ml of nanoparticles containing coumarin 6. 7-Aminoactinomycin D (7-AAD) was used as a marker for cell death. Flow cytometric analysis of the cells was performed at different time intervals to measure the fluorescence intensity of coumarin 6 and the percent of 7-AAD positive (dead) cells. (C–L) Visual observation of the nanoparticle loaded MSCs under fluorescent microscope (C–G) and phase-contrast microscope (H–L) at different time intervals after incubation with nanoparticles. (M) Role of P-gp in drug efflux from MSCs in vitro. MSCs were incubated with 0.25 μM calcein AM at 37 °C for 30 min in the presence or absence of 1 μM Zosuquidar. Data shown is mean ± S.D., n = 3, *P b 0.05. (N) In vitro drug release from nano-engineered MSCs. Nano-engineered MSCs were suspended in media containing 1% captisol and at different time points, the cells were centrifuged and the supernatant assayed for paclitaxel content. Data shown is mean ± S.D., n = 3.

their use as a carrier for small molecules has not been pursued, likely due to the difficulty in immobilizing small molecules in cells. Overexpression of drug efflux transporters such as P-glycoprotein in these cells further limits the loading of chemotherapeutic agents, many of which are substrates for efflux transporters (Fig. 2M) [10]. Previous studies have shown that nanoparticles formulated from the polyester polymer PLGA can be used as intracellular drug depots [22]. Following endocytic uptake, nanoparticles escape the endo-lysosomes and are retained in the cytoplasm for several days [23]. Further, studies have shown that the drug released intracellularly from nanoparticles is susceptible to efflux from cells [24]. Based on these observations, we hypothesized that PLGA nanoparticles can be used to load and release cytotoxic drugs like paclitaxel in MSCs. The hypothesized mechanism of action is illustrated in the Graphical abstract.

PLGA nanoparticles offer a number of advantages for use in nano engineering MSCs. Unlike the viral vectors, which are often utilized to transduce MSCs [25], PLGA nanoparticles are non-immunogenic and non-toxic [26]. Further, PLGA nanoparticles are biodegradable, capable of high drug loading efficiency, and can be tuned to achieve variable drug release rates [27]. A previous study demonstrated loading of poly-lactide nanoparticles containing imaging agents into MIAMI cells for brain targeting but no therapeutic agent was loaded in these particles [15]. However, that study provided the first proof-of-concept that MSCs can be loaded with polymeric nanoparticles. In our studies, we used PLGA nanoparticles to load paclitaxel, an anticancer cytotoxic drug used as a front line therapy for a number of solid tumors. Paclitaxel is susceptible to hydrolysis in aqueous systems such as phosphate buffered saline [28,29]. The hydrolytic products of

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Fig. 3. Cytotoxic potential of nano-engineered MSCs. (A) Long term survival of MSCs after nanoparticle loading. MSCs were incubated with paclitaxel nanoparticles or solution and assayed for cell survival (MTS assay) after 3 and 5 days of treatment. Data shown is mean ± S.D., n = 3. Cytotoxic potential of nanoengineered MSCs was determined in (B) A549 cells and (C) MA148 cells. Different cellular densities of nanoengineered MSCs were added on the top donor wells of a Transwell® plate having adherent tumor cells in the receiver chamber. MTS was performed on days 3 and 5 after treatment. Data shown is mean ± S.D., n = 3. (D) IC50 values for A549 and MA148 cells after 5 days. Data shown is mean ± S.D., n = 3. (E) Dose–response curves for cytotoxicity in A549 and MA148 cells 3 days after treatment with paclitaxel in solution or in nanoparticles. Data shown is mean ± S.D., n = 3, *P b 0.05 vs. control.

paclitaxel are difficult to quantify by standard analytical techniques. Further, the low aqueous solubility of the drug presents difficulties in achieving sink conditions without using large volumes of the release buffer [30]. A comparative study of different release medium for paclitaxel suggested that both the drug hydrolysis and improved solubility can be achieved by using cell culture media supplemented with 10% Captisol® (a beta-cyclodextrin analogue) [28]. Nanoparticles demonstrated an initial burst release over 1 h followed by steady release over 9 days. The initial burst is characteristic of polymeric nanoparticles encapsulating hydrophobic drugs and is attributed to the drug molecules present on or near the nanoparticle surface. It was interesting to note that the burst release occurred in the first 4 h. This is particularly advantageous since nano-engineering of MSCs typically requires between 4– 6 h during which the burst release from nanoparticles is complete and thus the particles loaded in the cells are in the sustained release phase. This would ensure that after systemic delivery of nanoengineered MSCs, the drug is released slowly and not dumped into the systemic circulation before the cells reach the tumor. Nanoparticle loading in cells is a dynamic process, governed by both endocytosis into and exocytosis out of the cells [31]. We initially optimized the conditions for nanoparticle loading in MSCs by evaluating

the effect of dose of nanoparticles and the duration of incubation. These studies suggested that the maximal uptake of nanoparticles occurred after incubation with 100 μg/ml of nanoparticles for 4–6 h. Similar to what has been reported earlier in other cells [32], nanoparticles were found co-localized in the lysosomes (yellow) and also present free in the cytosol (green) (Supplementary Fig. 4). While the goal was to achieve high loading of nanoparticles in MSCs, it was also critical to ensure that nanoparticle loading did not affect MSC viability or their native properties. Our studies show that loading paclitaxel–PLGA nanoparticles did not affect the short-term or long-term viability of MSCs. Additionally, nanoparticle uptake into the MSCs and their survival were similar in both adherent and suspended MSCs. This observation has important translational implication. Cultured MSCs can be loaded with nanoparticles in the tissue culture plate and then harvested for in vivo administration while freshly isolated MSCs in suspension can be treated immediately with nanoparticles without the necessity of plating them first. The differentiation potential of MSCs is commonly studied by monitoring their osteogenic, adipogenic and chondriogenic differentiation into bone, adipose and cartilaginous tissues, respectively [33]. Nanoengineered MSCs stained for lipid vacuoles and calcium deposits at

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Fig. 4. Differentiation and migration potentials of nano-engineered MSCs. Untreated MSCs (A, B) and nano-engineered MSCs (C, D) were grown in osteogenic and adipogenic differentiation media for 3 weeks following which the cells were fixed and stained with oil red O or alizarin red to detect lipid vacuoles or calcium deposits respectively. Control MSCs (E, F) were also stained similarly and used as negative control. (G) The migratory potential of MSCs towards serum gradient in a Transwell® plate. Untreated MSCs and nano-engineered MSCs were allowed to migrate from a serum free media towards complete growth media in a Transwell® plate. The migrated cells after 20 h were detached, stained with calcein AM and the percent cell migration was quantified. Data shown is mean ± S.D., n = 3.

the same extent as untreated MSCs, thus suggesting that nanoparticle loading does not affect their differentiation potential. A property critical to the use of MSCs as anticancer drug carriers is their migratory potential. In vitro testing of migration potential of cells is often performed in Transwell® plates, where the migration of cells towards a chemoattractant can be monitored and quantified [34]. In our studies, both untreated and nano-engineered MSCs migrated at similar rates towards 5% serum. Limited migration was observed in treatment wells lacking the chemoattractant, suggesting that MSC migration was highly specific and not due to the “leakage” of cells through the Transwell® pores. Potential for nanoparticle-loaded MSCs to induce tumor cell kill was studied in two different tumor cell lines. The ovarian tumor cell line

MA148 is sensitive to paclitaxel and showed substantial cell death in the presence of nano-engineered MSCs. Significant decrease in cell survival between 3 and 5 days at higher cell numbers suggests that nanoengineered MSCs continue to release the drug over this time frame, resulting in higher cell kill over time. Similar trend in cell kill was also observed in A549 lung tumor cells. Contrary to the effect of high nano-engineered MSC dose on prolonged cell kill, lower dose can lead to sub-optimal cell death; this is characterized by higher cell survival at 5 days after treatment in comparison to 3 days. These observations provide critical insights for further optimization of nano-engineered MSCs. Our studies show that paclitaxel release from nano-engineered MSCs was sustained. In the first 24 h, about 2.1% of the loaded drug

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Fig. 5. In vivo biodistribution of nano-engineered MSCs. In vivo tumoritropic properties of nanoengineered MSCs were studied in mice bearing orthotopic lung tumors. (A) Bioluminescent imaging of tumors in mice lungs on the day of injection. (B) Fluorescence imaging of mice at different intervals after treatment.

was released and is available for cancer cell kill (Fig. 2N). A comparison of the IC50 of free paclitaxel in solution with the number of nanoengineered MSCs required to achieve the same results allows for the determination of the ‘paclitaxel equivalent concentration (PEC)’ in the cells (PEC = IC50 solution / IC50 cells) [35]. According to that calculation, the PEC of nano-engineered MSC is between 0.075–0.23 pg/cell. Considering 4.7 pg of paclitaxel per cell, this number translates to about 1.6– 4.9% of the active paclitaxel released from the cells in 3 days. Thus, the predicted release rate matches with the observed release profile of paclitaxel from nano-engineered MSCs (Fig. 2N). Hence, the prolonged residence of drug loaded MSCs in the tumor environment [36] in combination with sustained drug release can be expected to translate into sustained and tumor-targeted delivery of paclitaxel, which otherwise gets cleared within a few hours after administration [37]. Tumor microenvironment is rich in pro-inflammatory cytokines, which act as chemoattractants for MSCs. MSCs possess surface markers such as SDF-1, IFN-γ, CCL5/CCR5, and CCR2 that allow them to home to sites of inflammation in vivo [38]. In vivo distribution of nanoengineered MSCs was compared with that of nanoparticles in an orthotopic lung tumor model. To facilitate in vivo imaging of nanoparticles and nano-engineered MSCs, a near-infrared dye (SDB 5491) was encapsulated in nanoparticles [39]. It has been previously reported that PLGA nanoparticles without any surface modification primarily accumulate in reticuloendothelial organs, liver and spleen [26]. In accordance with prior reports, we observed significant fluorescent signal from the abdominal region of the animals. Nano-engineered MSCs, on the other hand, appear to migrate directly to the lungs. Fluorescence signal was detectable in the thoracic area for 24 to 48 h, beyond which the fluorescence signal was below detectable limits. It is possible that slow release of the hydrophobic dye from nanoparticles resulted in the loss of fluorescence signal beyond 48 h.

The in vivo fate of MSCs after intravenous injection has previously been studied. In a systematic study, Kidd et al. showed that following intravenous injection in healthy mice, MSCs migrated to the lung and then to the liver and spleen [36]. This might partly be due to the high vascularity of the lung, liver and spleen. The migratory pattern of the MSCs differed in a wound model, where following initial residence in the lungs, MSCs migrated to the wound sites between 3 and 5 days. In mice bearing MDA-MB-231 metastatic tumors, intravenously injected MSCs demonstrated tropism for lung metastasis. In another study, intraperitoneally injected MSCs migrated and resided at sites of HEY ovarian tumor while intravenously injected MSCs trafficked to subcutaneous 4T1 tumors [36]. While additional long-term efficacy studies are needed, our current studies point to the potential of nano-engineered MSCs as tumortargeted carriers for the delivery of small molecules. Future studies will examine the long-term fate of nano-engineered MSCs as well as their anticancer efficacy in orthotopic tumor models. 5. Conclusions MSCs were nano-engineered using PLGA nanoparticles to carry high drug payload without significantly affecting their viability. Key properties of MSC such as their differentiation and migration potentials were unaffected by nano-engineering. MSCs carrying paclitaxel were effective in killing A549 lung cancer and MA148 ovarian cancer cells in vitro in a dose dependent manner. In vivo body-distribution studies showed that nanoparticles deposited preferably in the liver and spleen, while nano-engineered MSCs resided predominantly in the lungs for 24 h after injection. These studies demonstrate the potential of nanoengineered MSCs as a tumor-targeted delivery system for anti-cancer therapeutics.

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