Development of Exosome-encapsulated Paclitaxel to Overcome MDR in Cancer cells

Cells

RAW 264.7 macrophages, Madin-Darby canine kidney MDCK_WT and MDCK_MDR1 cells were purchased from ATCC (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco) supplemented with 10% FBS, 1% penicillin and streptomycin at 37°C and 5% CO₂. Murine Lewis lung carcinoma cell subline (3LL-M27), a highly metastatic lung clone, was a generous gift from Dr. L. Pelletier (CHUL, Laval University, QC, Canada).

Pgp protein levels in sensitive and resistant cancer cells were determined by western blot as previously reported (28) using monoclonal antibodies to Pgp, C219 (Dako Corp., Carpinteria, CA; at dilution 1:100), and secondary horseradish peroxide donkey anti-mouse IgG antibodies (Amersham Life Sciences, Cleveland, OH; at dilution 1:1500). To correct for loading differences, the Pgp levels were normalized to the constitutively expressed β-actin stained with anti-β-1-chicken integrin (Sigma Chemical Co., at dilution 1:200). Specific bands were visualized using a chemiluminescence kit (Pierce, Rockford, IL).

Characterization of Exosomes

Exosomes were harvested from the conditioned media of RAW 264.7 cells cultured in exosome-depleted media using the ExoQuick-TC™ Kit (System BioSciences; Mountain View, CA) and characterized by Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM) and Western Blot Analysis as described previously (29).

BODIPY-PC was used as a probe to examine the fluidic properties of exosomal membranes as described earlier (30). Briefly, 30 μL exosomes with a concentration of 4×10¹¹ particles/mL were mixed with 20 μL BODIPY-PC (0.03 mg/ml) (31) and supplemented with 70 μL deionized water; the mixture was incubated for 45 min at 37°C in the dark. Unbound label was removed using a Zeba^TM column (Life Technologies).

Drug Loading into Exosomes

For PTX and DOX loading into exosomes, purified exosomes (~10¹¹ exosomes) were first mixed with PTX or DOX in 1 mL PBS. Different methods of drug loading were investigated: incubation at room temperature (RT), electroporation, and sonication. For the incubation method, the admixture was incubated at 37°C for 1 hour with shaking. For the electroporation method, exosomes were mixed with PTX and added to a chilled 4 mm electroporation cuvette. The mixture was then electroporated using an Eppendorf Eporator (Eppendorf AG, Hamburg, Germany) at 1000 kV for 5 ms, and then incubated at 37°C for 30 min to allow for recovery of the exosomal membrane. For the sonication method, the PTX-exosome or DOX-exosome mixture was sonicated using a Model 505 Sonic Dismembrator with .25” tip with the following settings: 20% amplitude, 6 cycles of 30 s on/off for three minutes with a two minute cooling period between each cycle. After sonication, exoPTX or exoDOX solution were incubated at 37°C for 60 min to allow for recovery of the exosomal membrane. Excess free drug was separated from exoPTX or exoDOX by size exclusion chromatography using a NAP-10 Sephadex G25 column (GE Healthcare, Buckinghamshire, UK).

The amount of PTX loaded into exosomes was measured by a high performance liquid chromatography (HPLC) method. Briefly, exoPTX (10¹⁰ exosomes/0.1mL) in a microcentrifuge tube was placed on a heating block set to 75°C to evaporate solvent. Then, an equal volume of acetonitrile was added and the mixture was vortexed, sonicated and then centrifuged at 13,000 rpm (Thermo Legend Micro 21) for 10 min. Following centrifugation, the supernatant was taken and filtered through a Corning Regenerated Cellulose .2 μm syringe filter and transferred into HPLC autosampler vials. 20 μL aliquots were injected into the HPLC system (Agilent 1200, Agilent Technologies, Palo Alto, CA). All analyses were performed using a C18 column (Supelco Nucleosil C18, 250 mm × 4.6 mm, 5 μm, 100 Å, Sigma-Aldrich,) with a mobile phase of H₂O:acetonitrile (45:55, v/v) at a flow rate of 1 mL/min at 30°C. Absorbance was measured at 227 nm to monitor the elution of PTX.

To measure PTX release, freshly prepared exoPTX were placed in a 300K MWCO Float-A-Lyzer G2 device (Spectrum Laboratories, Houston, TX). The device was then placed in PBS under sink conditions at RT with stirring. Samples were taken at time points from inside the dialysis tube and were analyzed by HPLC as described above. The amount of PTX released from exoPTX was expressed as a percentage of total PTX and plotted as a function of time.

Accumulation of Exosomes and Exosome-incorporated PTX in Cancer Cells

To quantify the amount of exosomes taken up by cells, exosomes were stained with a lipophilic fluorescent dye, DIL as described previously (29). Then, DIL-labeled exosomes, or fluorescently-labeled liposomes, or polystyrene nanoparticles (NPs, Fluoro-Max G100, Thermo Fisher Scientific), were added in equal numbers (~10⁸ particles/well) and incubated with 3LL-M27 cells at 37°C and 5% CO₂ for various times. After each time point, the media was removed and cells were washed 3x with PBS and fixed by incubating with Formal-Fixx (Thermo Fisher Scientific), and examined by confocal microscopy or using a Shimadzu RF5000 fluorescent spectrophotometer. In case of exoPTX or Taxol, drugs were added in equimolar amounts to the MDCK_WT, or MDCK_MDR1 cells and incubated for 72h. The cell suspension was then lysed and analyzed for PTX content by HPLC as described above.

In vitro Cytotoxicity Assay

The in vitro antitumor efficacy of exoPTX was assessed using a standard MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide) assay with three cancer cell lines, and compared to Taxol as described earlier (32). Briefly, cancer cells (MDCKMDR1, MDCKwt, and 3LL-M27) were seeded at 5,000 cells/well in 100 μL of media in 96-well plates overnight. Various concentrations of exosomes isolated from macrophages conditioned media and loaded with PTX by sonication, or empty sonicated exosomes, or Taxol, or PTX were added to cancer cells for 48 hours at 37C, 5% CO2. Following the incubation, the cells were washed and incubated with MTT reagent as described in (33). The cytotoxic activity of PTX was then evaluated using a standard MTT assay (34). The absorbance at 570nm was measured using a Shimadzu RF5000 fluorescent spectrophotometer. The survival values were determined in relation to control cells cultured in drug-free media. All experiments were repeated at least three times. SEM values were less than 10%.

Production of a Lentiviral Vector (LV) and Transduction of LLC cells

Lentiviral vector encoding a fusion between the optical reporter mCherry (GBM8FlmC, red) and firefly luciferase (FLuc) were created by PCR amplification of the cDNA sequences for mCherry and FLuc from pEmCherry (Clontech) and pcDNA-Luciferase (Addgene) with restriction enzyme sequences that were engineered into the primers. To create the final constructs, mCherry was digested with BamHI/EcoV and FLuc was digested with EcoV/XhoI. The digested fragments were ligated into the BamHI/XhoI digested pTK402 LV transfer vector (a kind gift from Dr. Tal Kafri, The University of North Carolina at Chapel Hill). LV-mCherryFLuc viral vectors were packaged in 3LL-M27 cells by transient transfection using the psPAX2 and pMD2.G (Addgene) packaging plasmids and following previously described protocols (35).

To utilize bioluminescence and fluorescence imaging, 3LL-M27 cells were transduced with lentiviral vectors encoding an mCherry and Renilla luciferase (mC-RL) fusion protein. The viral construct also encoded for a puromycin resistance gene downstream of mCherry which was introduced to enable for selection of nearly 100% positively transduced cells. A robust expression of both the fluorescent and bioluminescent markers was observed, and no difference in proliferation was detected between modified and unmodified cells. These cells (8FlmC-FLuc-3LL-M27) were used for biodistribution and therapeutic efficacy studies.

Biodistribution of Exosomes in Mice with Pulmonary Metastases

The experiments were performed with female C57BL/6 mice (Charles River Laboratories, Durham, NC) eight weeks of age in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animals were kept five per cage with an air filter cover under light- (12-hours light/dark cycle) and in a temperature-controlled (22 ± 1° C) environment. All manipulations with the animals were performed under a sterilized laminar hood. Food and water were given ad libitum.

C57BL/6 mice (n = 4) were injected intra tail vein (i.v.) with 8FlmC-FLuc-3LL-M27 cells (5×10⁶ cells/mouse in 100 μL saline) and tumor lung metastases were allowed to establish for 10-12 days. Twelve days following cancer cells i.v. injection, DID-labeled exosomes isolated from autologous macrophages were administered intranasally (i.n., 10⁷ particles/10μl × 2) to mice with lung metastases. Four hours later, mice were sacrificed, perfused, lungs were extracted and sectioned on a microtome at a thickness of 20 μm; nuclei were stained with DAPI (300 mM, 5 min). The images of lung sections were examined by a confocal fluorescence microscopic system ACAS-570 and corresponding filter set, and processed using ImageJ software.

In another experiment, mice with established GBM8FlmC-metastases were injected i.n. with non-labeled exosomes loaded with Dox by sonication as described above (10⁷ particles/10μL × 2). Four hours later mice were sacrificed, perfused; lungs were extracted, sectioned, and co-localization of Dox with pulmonary metastases was visualized by confocal microscopy.

Therapeutic Efficacy of exoPTX against Pulmonary Metastases

The antineoplastic effects of exoPTX were evaluated in a mouse model of pulmonary metastases. For this purpose, C57BL/6 mice were i.v. injected with 8FlmC-FLuc-3LL-M27 cancer cells (5×10⁶ cells/100 μL/ mouse). Forty eight hours later, mice were treated i.n. with exoPTX (10⁷ particles/10 μl × 2), or Taxol (50 mg/kg/mouse), or saline as a control (n = 7) every other day with a total of seven treatments. Tumor progression was monitored by luminescence using the IVIS system as described in (36). The animals were imaged at various time points (1 – 22 days) post-treatment as described (36). The chemoluminescent signal was quantified by Living Image® 2.50 software. To assess the amount of cancer metastases at day 22, mice were sacrificed, perfused, and lung slides obtained on microtome (Thermo Scientific) were examined by confocal microscopy.

Accumulation and therapeutic efficacy of exoPTX in target cancer cells in vitro

The ability to deliver the drug payload into target cells was studied with fluorescently-labeled exosomes in 3LL-M27 cells, and compared to the commonly used nanocarriers, liposomes and polystyrene nanoparticles (NPs) (37) with the same size and level of fluorescence (Fig. 2). Liposomes were prepared by a reverse phase evaporation method as described previously (29). Confocal images revealed a profound accumulation of exosomes in cancer cells and limited uptake of liposomes and NPs (Fig. 2A). This result was further confirmed and quantitated in accumulation studies (Fig. 2B). Exosomes were taken up about 30 times better than the synthetic nanoparticles, suggesting that PTX loaded into exosomes can be efficiently delivered to cancer cells in therapeutically sufficient quantities. These results clearly show the advantages of exosome-based drug delivery systems over common synthetic nanocarriers and confirmed our previous report regarding the profound accumulation of exosomes in neuronal PC12 cells (29).

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Figure 2

A profound accumulation of exosomes in 3LL-M27 cells in vitro

3LL-M27 cells were incubated with fluorescently-labeled (red) exosomes, or liposomes, or PS NPs for various times and the amount of accumulated nanocarriers was examined by confocal microscopy (A), and spectrophotometry (B). Bar: 10 μm.

The anticancer effects of exoPTX were evaluated in a resistant MDR cells expressing the drug efflux transporter, Pgp (MDCK_MDR1), and their sensitive counterparts (MDCK_WT). The loading of PTX into exosomes significantly increased drug cytotoxicity as compared to PTX alone, or Taxol in both sensitive MDCK_WT and resistant MDCK_MDR1 cancer cells (Table 1). These results are consistent with earlier reports regarding increased cytotoxicity of another anticancer agent, Dox in cancer cells (23). The most intriguing observation was made, when the effects of various PTX formulations were compared in sensitive and resistant cancer cells. For this purpose, the increased cytotoxicity of the drug was expressed in the form of a “Resistance Reversion Index” (RRI), i.e. ratio of IC₅₀ of PTX alone, and in nanoformulation (e.g. IC_50,PTX/IC_50,exoPTX, or IC_50,PTX/IC_50,taxol). Both PTX formulations caused significant sensitization of MDR cells with respect to PTX (Table 1). In particular, RRI for exoPTX in MDCK_MDR1 and MDCK_WT was 53.33 and 18.38, respectively. In contrast, RRI for Taxol in both resistant and sensitive cancer cells was c.a. 6 (Table 1). Noteworthy, empty sonicated exosomes did not show any cytotoxicity in all studied cell lines (Supplemental Fig. 3). Thus, the increase in PTX cytotoxicity afforded by exoPTX was greater in Pgp-overexpressing cells than their sensitive counterparts (Table 1).

Mechanistic studies of exoPTX cytotoxic effects

We hypothesized that exoPTX may alter drug intracellular trafficking and bypass the drug efflux system more efficiently than Taxol (in particular, exoPTX may facilitate endosomal release of PTX from exosomes in cancer cells). To prove this hypothesis, we examined the accumulation levels of a fluorescent probe and Pgp substrate, Dox, incorporated into exosomes (exoDox) in MDCK_MDR1 and MDCK_WT cells. First, elevated Pgp expression levels in MDCK_MDR1 cells, and low, if any, Pgp levels in MDCK_WT cells were confirmed by western blot (Fig. 3A). Next, the uptake of free Dox and exosome-incorporated drug, exoDox, was compared in the presence/absence of a Pgp inhibitor, verapamil. As expected, the incorporation of Dox into exosomes significantly increased drug accumulation levels in both sensitive and resistant cancer cells (Fig. 3B). Inhibition of Pgp-mediated drug efflux by verapamil increased accumulation of free Dox in resistant MDCK_MDR1 cells, but did not alter drug accumulation in their sensitive counterparts. Remarkably, verapamil treatment did not affect exoDox accumulation in resistant MDCK_MDR1 cells, indicating that drug incorporation into exosomes allowed it to bypass this resistance mechanism (Fig. 3B).

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Figure 3

Effect of Pgp inhibition on Dox accumulation in MDR and sensitive cancer cells

The accumulation of free Dox or exoDox in MDCK_MDR1 and MDCK_WT cells was studied in cell lysates. The Dox incorporation into exosomes significantly increased accumulation in sensitive and resistant cells, while no effect of verapamil on exoDOX accumulation was found in both cell lines.

We demonstrated earlier that the incorporation of Pgp substrates, such as R123 or Dox, into block-copolymer-based nanocarriers, i.e. Pluronic micelles, increased drug accumulation in resistant cancer cells due to the inhibition of Pgp efflux transporter by Pluronic macromolecules incorporated into the membranes of resistant cancer cells (28, 38-40). To exclude the possibility that exosomes may inhibit Pgp-mediated efflux by their fusion with cellular membranes, accumulation of R123 in both resistant and sensitive MDCK cancer cells was assessed. R123 does not incorporate into exosomes upon incubation at RT, as was confirmed in our preliminary studies (Supplemental Fig. 4). For this purpose, MDCK_WT and MDCK_MDR1 cell monolayers were pretreated with a Pgp inhibitor, verapamil (positive control), or empty exosomes, or media (negative control), and then were treated with R123 solutions for two hours (Supplemental Fig. 5). R123 accumulation levels in resistant MDCK_MDR1 cells were increased almost five times in verapamil pre-treated cells. In contrast, treatment with empty exosomes did not affect R123 accumulation in MDCK_MDR1 cells (Supplemental Fig. 5). As expected, neither treatment with verapamil, nor with empty exosomes, altered R123 accumulation levels in sensitive MDCK_WT cells. This indicates that exosomes themselves do not appear to have any inhibitory effect on Pgp-mediated efflux; they allow incorporated drugs to bypass the Pgp efflux protein perhaps, through endocytosis-mediated transport and/or fusion with plasma membranes.

Co-localization of Airway-delivered Exosomes with Pulmonary Metastases in Lewis Lung Carcinoma (LLC) mouse model

To establish an in vivo model of pulmonary metastases, C57BL/6 mice were injected intra-tail vein (5×10⁶ cells/100 μL) with 3LL-M27 cells. Important, this model is particularly relevant to the present investigation, as it was demonstrated that 3LL-M27 tumor cells have high expression levels of the MDR1 gene and Pgp expression in vivo (32). Twenty days later, mice were sacrificed, perfused, and lungs were isolated, sectioned, and stained with Hematoxylin and Eosin (H&E). Multiple metastases were detected in whole lungs (Supplemental Fig. 6A). Histological evaluations revealed that the structure of alveoli in tumor-bearing lungs was disrupted by tumor cells (Supplemental Fig. 6B). Next, mice were injected with 8FlmC-FLuc-3LL-M27 (red, Fig. 4) intra-tail vein as described in Materials and Methods section. 22 days later, autologous exosomes stained with a fluorescent dye, DiD (green), were i.n. administered to mice with pulmonary metastases. Four hours later, mice were sacrificed, perfused; lungs were sectioned on microtome and examined by confocal microscopy. Nuclei were stained with DAPI (blue, Fig. 4). Confocal images revealed 97.9 ± 2.0% of exosomes were co-localized with lung metastases (Fig.4), indicating efficient targeting of exoPTX in vivo. A similar experiment was performed with exoDox formulation in order to visualize drug delivery to pulmonary metastases (Supplemental Fig. 7). Non-labeled exosomes loaded with Dox (green, Supplemental Fig. 7B) were i.n. administered to mice with established 8FlmC-FLuc-3LL-M27 metastases (red, Supplemental Fig. 7A). Confocal images revealed a substantial amount of DOX in the lungs co-localized with cancer cells (yellow, Supplemental Fig. 7C). These results indicate that airway-administered exosomes reached pulmonary metastases and delivered their drug payload to target cancer cells.

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Figure 4

Co-localization of airway-delivered exosomes with pulmonary metastases

Exosomes were isolated from macrophages conditioned media, and labeled with fluorescent dye, DID (green). C57BL/6 mice were i.v. injected with 3LL-M27 cells transduced with lentiviral vectors encoding the optical reporter mCherry (8FlmC) fluorescent protein. 21 days later, the mice with established pulmonary metastases (red) were i.n. injected with DID-labeled exosomes (green). 4 hours later, mice were euthanized, perfused, lungs were sectioned, and stained with DAPI (blue). The confocal images revealed near complete co-localization of exosomes with metastases (yellow). Images were obtained with ×10 (A), and ×60 (B) magnification. Bar: 50 μm.

This study was supported by the United States National Institutes of Health grants 1RO1 NS057748 and The Carolina Partnership, a strategic partnership between the UNC Eshelman School of Pharmacy and The University Cancer Research Fund through the Lineberger Comprehensive Cancer Center (to EVB), RR021937 (to AVK), and Ministry of Education and Science of Russian Federation grants 11.G34.31.0004, 02.740.11.5232, and RSF-14-13-00731 (both to AVK and NLK).