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, 12 (3), 655-664

Development of Exosome-Encapsulated Paclitaxel to Overcome MDR in Cancer Cells


Development of Exosome-Encapsulated Paclitaxel to Overcome MDR in Cancer Cells

Myung Soo Kim et al. Nanomedicine.


Exosomes have recently come into focus as "natural nanoparticles" for use as drug delivery vehicles. Our objective was to assess the feasibility of an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat MDR cancer. Herein, we developed different methods of loading exosomes released by macrophages with PTX (exoPTX), and characterized their size, stability, drug release, and in vitro antitumor efficacy. Reformation of the exosomal membrane upon sonication resulted in high loading efficiency and sustained drug release. Importantly, incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCKMDR1 (Pgp+) cells. Next, our studies demonstrated a nearly complete co-localization of airway-delivered exosomes with cancer cells in a model of murine Lewis lung carcinoma pulmonary metastases, and a potent anticancer effect in this mouse model. We conclude that exoPTX holds significant potential for the delivery of various chemotherapeutics to treat drug resistant cancers.

From the clinical editor: Exosomes are membrane-derived natural vesicles of ~40 - 200 nm size. They have been under extensive research as novel drug delivery vehicles. In this article, the authors developed exosome-based system to carry formulation of PTX and showed efficacy in the treatment of multi-drug resistant cancer cells. This novel system may be further developed to carry other chemotherapeutic agents in the future.

Keywords: Cancer; Drug resistance; Exosome; Paclitaxel; Pgp.


Figure 1
Figure 1. Characterization of PTX exosomal formulations
Exosomes were collected from conditioned media of RAW 264.7 macrophages, and loaded with PTX by various methods: co-incubation at RT; electroporation, and sonication. The size of exoPTX was measured by NTA and DLS (A). The loading with PTX increased the size of exosomes, but did not significantly altered their surface charge. The loading efficiency of exosomes with PTX increased in a row: incubation at RT < electroporation << sonication. The exosome protein content was confirmed by western blot (B). Significant amount of exosome-associated proteins, Alix, TSG101, and Flotillin was detected in naïve (2) and sonicated exosomes (3), but not in the cells (1). Effect of sonication on fluidity of exosomal membranes labeled with BODIPY-PC was examined by fluorescence polarization measurements (C). The microviscosity of exosomal membranes was significantly decreased by six cycles of ultrasound treatment (3) compared to naïve exosomes (1), or exosomes subjected to one sonication cycle (2). The microviscosity of sonicated exosomes was completely restored following one hour incubation period at 37 (5), but not after 30 min incubation (4). The morphology of drug-loaded exosomes was examined by AFM (D). Images revealed small spherical naïve exosomes as well as PTX-loaded exosomes. The bar: 200 nm. A release PTX profile from pre-loaded exosomes was evaluated for the exoPTX formulation obtained by sonication (E). Values are means ± SEM (n = 4). Symbols indicate the relative level of significance compared with naïve exosomes (p < 0.05)
Figure 2
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.
Figure 3
Figure 3. Effect of Pgp inhibition on Dox accumulation in MDR and sensitive cancer cells
The accumulation of free Dox or exoDox in MDCKMDR1 and MDCKWT 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.
Figure 4
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.
Figure 5
Figure 5. The inhibition of metastases growth in mouse lungs upon exoPTX treatment
C57Bl/6 mice were i.v. injected with 8FlmC-FLuc-3LL-M27 (red) cells to establish pulmonary metastases. 48 hour later mice were treated with exoPTX, or Taxol, or saline, or empty sonicated exosomes as a control, and the treatment was repeated every other day, totally seven times. Representative IVIS images were taken at day 21 (A). Statistical significance of metastases levels from IVIS images in lungs of treated animals compared to control mice is shown by asterisk (*p < 0.05; **p < 0.005) (B). At the endpoint, 21 days later, mice were sacrificed, perfused, and lung slides were examined by confocal microscopy (C). The bar: 10 μm.

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