Engineering Macrophage-derived Exosomes for Targeted Paclitaxel Delivery

to Pulmonary Metastases: in vitro and in vivo Evaluations

Myung Soo Kim, Matthew J. Haney, Yuling Zhao, Dongfen Yuan, Irina
Deygen, Natalia L. Klyachko, Alexander V. Kabanov, Elena V. Batrakova

PII: S1549-9634(17)30178-8
DOI: doi: 10.1016/j.nano.2017.09.011
Reference: NANO 1669

To appear in: Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: 20 April 2017

Revised date: 11 September 2017
Accepted date: 19 September 2017

Please cite this article as: Kim Myung Soo, Haney Matthew J., Zhao Yuling, Yuan
Dongfen, Deygen Irina, Klyachko Natalia L., Kabanov Alexander V., Batrakova Elena
V., Engineering Macrophage-derived Exosomes for Targeted Paclitaxel Delivery to Pul-
monary Metastases: in vitro and in vivo Evaluations, Nanomedicine: Nanotechnology, Biol-
ogy, and Medicine (2017), doi: 10.1016/j.nano.2017.09.011

This is a PDF file of an unedited manuscript that has been accepted for publication.
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Engineering Macrophage-derived Exosomes for Targeted Paclitaxel Delivery

to Pulmonary Metastases: in vitro and in vivo Evaluations

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Myung Soo Kim1,2, Matthew J. Haney1,2, Yuling Zhao1,2, Dongfen Yuan1,2, Irina Deygen3,
Natalia L. Klyachko1,2,3, Alexander V. Kabanov1,2,3, and Elena V. Batrakova1,2,*

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Center for Nanotechnology in Drug Delivery, 2Eshelman School of Pharmacy, University of

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North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA, and 3Deparment of Chemical
Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia
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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
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Comprehensive Cancer Center (to EVB), RR021937 (to AVK), and grant RSF-14-13-00731
(both to AVK and NLK).
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Abstract word count: 147

Complete manuscript word count: 5,001
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Number of references: 40
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Number of figures: 6
Number of Tables: 0
Number of Supplementary online-only files: 1

Running title: Paclitaxel-loaded exosomes targeted to pulmonary metastases

*Correspondence should be addressed to E.V.B. (batrakov@ad.unc.edu)

UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599-7362, Phone: 919-537-3712, Email: batrakov@email.unc.edu

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ABSTRACT

Exosomes have recently emerged as a promising drug delivery system with low immunogenicity,

high biocompatibility, and high efficacy of delivery. We demonstrated earlier that macrophage-

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derived exosomes (exo) loaded with a potent anticancer agent paclitaxel (PTX) represent a novel

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nanoformulation (exoPTX) that shows high anticancer efficacy in a mouse model of pulmonary

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metastases. We now report the manufacture of targeted exosome-based formulations with

superior structure and therapeutic indices for systemic administration. Herein, we developed and

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optimized a formulation of PTX-loaded exosomes with incorporated aminoethylanisamide-
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polyethylene glycol (AA-PEG) vector moiety to target the sigma receptor, which is

overexpressed by lung cancer cells. The AA-PEG-vectorized exosomes loaded with PTX (AA-
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PEG-exoPTX) possessed a high loading capacity, profound ability to accumulate in cancer cells

upon systemic administration, and improved therapeutic outcomes. The combination of targeting
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ability with the biocompatibility of exosome-based drug formulations offers a powerful and
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novel delivery platform for anticancer therapy.

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Words: 147

Key words: drug delivery systems, exosomes, paclitaxel, pulmonary metastases.

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1. INTRODUCTION

The lung is one of the most common sites of metastases and tumor relapse following treatment of

the primary tumor mass, lung metastases are identified in 30 – 55% of all cancer patients.

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Because pulmonary metastases are distributed throughout the pulmonary parenchyma, their

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excision is difficult, if not impossible. Clinical reports have highlighted a rare occurrence of

complete regression for metastatic tumors in patients who underwent standard-of-care palliative

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treatment involving radiation or immunotherapy. As a result, lung cancer is the leading cause of

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cancer-related deaths in the world, responsible for more deaths than breast, colon, and prostate

cancers combined (1). In particular, non-small cell lung cancer (NSCLC) is responsible for
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~85% of all lung cancers, and prognosis of metastatic NSCLC is poor; chemotherapy provides

only minimal increases in survival rates, with a five-year survival rate of less than 15% (2, 3).
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Thus, the efficient, targeted delivery of anticancer agents to pulmonary metastases remains one
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of the greatest challenges for the therapy.

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The recently emerged field of nanotechnology holds great promise for developing drug delivery

systems with targeting and controlled-release characteristics for cancer treatment; there have
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been many new advances and innovations made in this field during the past decade (1). A large

proportion of chemotherapeutic drugs have low aqueous solubility, consequently requiring the

use of specialized delivery vehicles (e.g. micelles, liposomes, polymeric nanoparticles, or other

types of nanoparticles) for parenteral administration. However, these nanosized delivery vehicles

are often difficult to manufacture and may cause unwanted side effects (such as the excipient

Cremophor EL in the commercial formulation of PTX, Taxol). In addition, conventional drug

nanoformulations normally are cleared rapidly from the circulation by the mononuclear

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phagocyte system (MPS) (4). This demands innovative approaches for engineering drug delivery

systems.

Exosomes are membrane-derived vesicles ~ 40 - 200 nm in diameter (5); they may be found in

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extracellular bodily fluids and in conditioned cell culture media (6). Exosomes are released by a

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variety of cell types; and formed when multi-vesicular bodies inside the cells fuse with the

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plasma membrane and release intraluminal vesicles into the extracellular microenvironment (6,

7). Exosomes naturally function as intracellular messengers, carrying RNAs and proteins

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between the cells (8). Recently, exosomes have begun to be explored for use as drug delivery
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vehicles for non-native therapeutics such as nucleic acids (9-14), therapeutic proteins (15), and

small molecule drugs (6, 16, 17). The membranotropic nature of exosomes suggests that these
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drug carriers will be able efficiently interact with the target cancer cells and deliver their toxic

payload. This process is facilitated by membrane interactions and fusion due to the expression of
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various adhesive proteins (tetraspanins and integrins) on the surface of exosomes (18).
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Noteworthy, it was reported that exosomes are able to deliver their intraluminal cargo into the

cytosol of target cells (19). In addition, allogenic exosomes have an immune privileged status,
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which allows for decreased drug clearance by MPS. Specifically, immunocytes-derived

exosomes are known to express CD47 receptor (20), which interacts with signal regulatory

protein α (SIRPα) to produce a “don’t eat me” signal in phagocytes (21). These unique features

make exosomes an attractive option for use as a drug delivery vehicle for cancer treatment.

We reported earlier that macrophage-derived exosomes can be loaded with low molecular

chemotherapeutics, such as PTX, and doxorubicin (DOX) (22), or therapeutic proteins, such as

catalase (15) or brain derived neurotrophic factor, BDNF (23). Different loading procedures (co-

incubation with a drug at room temperature, electroporation, saponin permeabilization, extrusion,

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freeze-thaw cycles, or sonication) were utilized to incorporate these therapeutic agents into

exosomes. We demonstrated that a reformation of exosomal membranes upon sonication allowed

efficient drug loading along with preservation of exosomal structure and internal content. Here

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we report the development of improved exosome-based PTX formulation targeted to cancer cells

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for systemic administration. It has been shown that a variety of cancer types, including NSCLC,

overexpress sigma receptor (24), a membrane-bound protein with an as-yet undefined role. AA

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is a ligand with high affinity for sigma receptor and has been utilized to targeted delivery of Dox,

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proteins, and siRNA (25-27). Next, the most common method of reducing the immunogenicity of

nanoformulated drugs is to decorate the nanoparticle in a polyethylene glycol (PEG) corona,

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which reduces recognition by the MPS and aids in avoiding clearance. To this end, Kooijmans et

al. have recently shown that the introduction of polyethylene glycol to exosomes results in
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stealth properties, which significantly increases their circulation time in mice (28).
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Based on these findings, we hypothesized that modification of PTX-loaded exosomes with PEG-
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AA vector moiety will improve their circulation time in the blood and allow to target pulmonary

metastases. Our investigations revealed a robust accumulation and nearly complete co-
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localization of systemically administered AA-exosomes with pulmonary metastases, and greater

in vivo therapeutic efficacy as compared to non-vectorized exoPTX and Taxol. Thus, exosome-

based formulations may represent the next generation of drug delivery systems that combines

nanoparticle size with targeted drug delivery and low immunogenic profile.

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2. MATERIALS AND METHODS

Description of reagents, cell culture conditions, animals used in these studies, characterization of

exosomal formulations as well as accumulation and intracellular trafficking experiments and

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statistical analysis are described in Supplementary Material.

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2.1 Preparation of AA-Vectorized Exosomes Targeted to the Sigma Receptor

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For all in vitro experiments, exosomes were harvested from the supernatants of RAW 264.7 cells

cultured in exosome-depleted media using the ExoQuick-TC™ Kit as described earlier (22). For

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all in vivo experiments, exosomes released by primary bone-marrow derived macrophages
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(BMM) isolated from C57BL/6 mice were utilized. Exosomes targeted to sigma receptor with

DSPE-PEG-AA (AA-PEG-exo) and non-vectorized control exosomes with DSPE-PEG (PEG-

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exo) were prepared as follows: exosomes were isolated from macrophage media as described

above and then DSPE-PEG or DSPE-PEG-AA (50 µg/ml) were added to the mixture (for PEG-
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exo and AA-PEGexo, respectively). 100μL PTX (10mg/mL in ethanol) was also added to the
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exosomes to incorporate PTX using sonication method that was developed in our laboratory (22).

After sonication, AA-PEGexo, or PEGexo, or AA-exoPTX solutions were incubated at 37° C for
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60 min to allow for recovery of the exosomal membrane. Exosomes were purified from the

excess of free DSPE-PEG or DSPE-PEG-AA by size exclusion chromatography using a NAP-10

Sephadex G25 column (GE Healthcare, Buckinghamshire, UK). Purified exosomal formulations

were collected and stored at -20° C.

2.2 Drug Loading and Optimization AA-PEG-exoPTX

For PTX loading into vectorized exosomes, 1mL of purified exosomes (~1011 exosomes) in PBS

was mixed with PTX and DSPE-PEG-AA. For this purpose, first PTX (10 mg/mL drug in EtOH

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stock solution) was added to 1 mL exosomes in PBS. Then, different amounts of AA-PEG-

DSPE (0.05-0.50 mg/ml) in PBS were added to the mixture of exosome with PTX. The obtained

mixture was sonicated using a Model 505 Sonic Dismembrator using .25” tip (Thermo Fisher

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Scientific, USA) as described earlier (22). After the sonication, a solution AA-vectorized

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exosomes loaded with PTX (AA-PEG-exoPTX) were incubated at 37° C for 60 minutes to allow

for recovery of the exosomal membrane. The excess free PTX and AA-PEG-DSPE was

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eliminated from AA-PEG-exoPTX by size exclusion chromatography as described elsewhere

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(22). The amount of PTX loaded into exosomes was measured by HPLC method (22). All

analyses were performed using a C18 column (Supelco Nucleosil C18, 250 mm x 4.6 mm, 5 µm,
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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. Loading capacity is expressed by µg protein of exosomes.

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2.3 Biodistribution of Intravenously Injected Exosomes in Mice with Pulmonary

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Metastases
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To utilize fluorescence imaging, 3LL-M27 cells were transduced with lentiviral vectors encoding

the optical reporter FITC (FITCFlmC, green) fluorescent protein as reported earlier (29). The
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viral construct also encoded for a puromycin resistance gene downstream of FITC, which was

introduced to enable for the selection of positively transduced cells. C57BL/6 mice were injected

intra tail vein (i.v.) with FITC-FLmC-3LL-M27 cells (5x106 cells/mouse in 100 µl saline) and

pulmonary metastases were allowed to establish. In parallel, exosomes isolated from autologous

macrophages conditioned media were stained with DiL (red) and vectorized to sigma receptor

with DSPE-PEG-AA. To stain exosomes with DiL, exosome pellet was rehydrated in 500 µL

PBS, supplemented with 1 mM stock solution DiL in DMSO (5 µL), and incubated for 20

minutes at RT. Following incubation, exosomes were isolated by PEG precipitation and
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rehydrated in 500 µL PBS. The fluorescently-labeled exosomes were additionally purified from

remaining non-incorporated DiL on NAP 10 column. Twelve days following cancer cells

injection, mice with established lung metastases were intravenously (i.v.) injected with

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autologous vectorized exosomes DiL-AA-PEG-exo, or non-vectorized exosomes DiL-exo as a

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control group (108 particles/100 µl, n = 4 per group). Four hours later, mice were sacrificed,

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perfused, lungs were extracted and sectioned at a thickness of 20 μm; nuclei were stained with

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DAPI (300 mM, 5 minutes). The images of lung and liver sections were examined by a confocal

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fluorescence microscopic system ACAS-570 (Meridian Instruments, Okimos, MI) with argon ion

laser and corresponding filter set. Digital images were obtained using the CCD camera
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(Photometrics) and Adobe Photoshop software. Quantification of immunostaining was

performed with ImageJ software, utilizing JACoP plugins to calculate Pearson’s co-localization
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coefficients (30). The comparison was performed in randomized order of the blinded experiment
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on 10-15 sets of images acquired with the same optical settings.

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2.4 Therapeutic Efficacy of AA-PEG-exoPTX Against Pulmonary Metastases

The antineoplastic effects of PTX exosome formulation were evaluated in a mouse model of
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pulmonary metastases with 3LL-M27 cells transduced with lentiviral vectors encoding the

optical reporter mCherry (GBM8FlmC, red) as described earlier (22). To establish pulmonary

metastases, C57BL/6 mice were i.v. injected with 8FlmC-3LL-M27 cancer cells (5x106 cells/100

µl/ mouse). Forty-eight hours later, mice were treated i.v. with autologous AA-PEG-exoPTX, or

exoPTX, or empty exosomes (exo) (4x1011 particles/100 µl, three times on day 1, 4, and 7; 0.5

mg/kg totally), or Taxol (same regiment as exosome-based formulations), or saline as a control

(n = 7). To assess amount of cancer metastases, two mice from each group were sacrificed on

day 18, perfused, and lung slides were examined by confocal microscopy. The rest of the mice (n
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= 5) were monitored daily for the signs of the reduced physical activity and the progression of

the tumor. The survive time of each mouse was recorded. To perform quantitative analysis, an

additional experiment with the same treatment groups of mice (n = 5) was carried out. On day 18

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mice were sacrificed, perfused, and lung slides were examined by confocal microscopy. The

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metastases area on the slides was calculated using ImageJ software. The comparison was

performed in randomized order of the blinded experiment on 10-15 sets of images acquired with

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the same optical settings.

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2.5 Statistical Analysis
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For the all experiments, data are presented as the mean ± S.E.M. Tests for significant differences

between the groups were performed using a t-test or one-way ANOVA with multiple
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comparisons (Fisher's pairwise comparisons) using GraphPad Prism 5.0 (GraphPad software,

San Diego, CA, USA). A minimum p value of 0.05 was chosen as the significance level.
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3. RESULTS
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3.1 Manufacture and Characterization of AA-PEG-exoPTX

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We demonstrated earlier that efficient loading of PTX can be achieved, when exosomes are

subjected to ultrasound treatment in the presence of the drug (22). Herein, we applied the same

approach for simultaneous loading of PTX, and vectorization of exosomes to sigma receptor

using AA moiety. Obtained formulations were characterized by size, charge, protein content, and

PTX loading capacity (LC). First, the effect of incorporation of vector lipid molecule (DSPE-

PEG-AA) on the LC was evaluated. LC was expressed as the amount of the drug vs. the amount

of exosomal protein. HPLC analysis revealed that incorporation of high amounts of AA-PEG-

DSPE (0.5 mg/ml) into exosomes significantly decreased their LC for PTX (Fig. 1 A). In

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contrast, lower amounts of the lipid (0.25 - 0.05 mg/ml) did not affect LC for PTX. It is likely

that excess of hydrophobic chains of the lipid incorporated into exosomal membranes diminished

available for PTX space. Based on these findings, the highest amount of the incorporated vector

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moiety (0.25 mg/ml) that did not significantly reduce PTX loading in exosomes was selected for

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all further evaluations. LC for the optimal AA-PEG-exoPTX formulation was ~33%, comparable

to the LC achieved for non-vectorized exoPTX (Fig. 1 A). Noteworthy, ultrasound treatment

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significantly increased amount of PTX incorporated into exosomes; the LC in exosomes without

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sonication was as low as 1.4% (22).
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To address a concern about possible alterations in the exosomal membranes upon incorporation

of vector moiety (AA-PEG-DSPE) using sonication, the levels of exosome-specific proteins,

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TSG101 and flotillin, in different exosomal formulations were examined by western blot (Fig. 1

B). The mild sonication utilized for PTX loading with six cycles, and intermediate time out for
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cooling down and membrane restoration did not affect the protein content of exosomes. In
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particular, sonicated vectorized and non-vectorized exosomes, as well as vectorized exosomes

loaded with PTX showed elevated expression of exosome-associated proteins (TSG101, and
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flotillin) as compared to cell lysate, which displayed greater levels of -actin (Fig. 1 B).

Noteworthy, we reported earlier that sonication itself did not affect levels of exosome-specific

proteins compared to naïve non-sonicated exosomes (22). Furthermore, exosomal formulations,

as well as parental macrophages, were also found to express the lymphocyte function associated

antigen-1 (LFA1, subunit CD11a) (Fig. 1 B), which assists in cell uptake and may bind to

endothelial cell adhesion molecules overexpressed on activated endothelial cells, such as those

found in tumors (31). This is important, since the presence of LFA1 on the surface may improve

specific targeting of exosome-based PTX formulations to cancer cells.

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Finally, the hydrodynamic size was determined by DLS and NTA (Fig. 1 C). Naïve empty

exosomes had a narrow size distribution, with an average particle diameter of 110.8 ± 4.1 nm

and 75.9 ± 2.6 nm as revealed by NTA and DLS, respectively. The sonication procedure

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significantly increased size of exosomes up to 291 ± 3.5 nm (Fig. 1 C). Noteworthy, exosomes

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sonicated in the presence of PTX were smaller than those sonicated without PTX. This effect

may be due to the stabilization of exosomal membranes by the incorporated drug. Next, it is

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known that sialic acid on glycosylated lipids and proteins is abundant on cell membranes and

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contributes to the surface charge of individual cellular membranes. In this regard, loading of

exosomes with PTX did not significantly alter the slightly negative change of the nanocarriers
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(Fig. 1 C). However, vectorized AA-PEG-exoPTX formulation were found to have a less

negative surface charge than exoPTX, probably due to the shielding of the exosomal membrane
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by the long PEG chains of the lipid (Fig. 1 C).

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3.2 Effect of AA-PEG-DSPE Incorporation on Membrane Fluidity in Exosomes

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Fluidity of exosomal membrane upon AA-PEG-DSPE incorporation was examined using

BODIPY-PC. This is a hydrophobic fluorescent compound, which incorporates in the

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hydrocarbon regions of lipid membranes. Transfer of BODIPY-PC from the aqueous

environment into lipid bilayers results in a drastic increase of the fluorescence emission for this

probe. Once the probe is incorporated into lipid membranes, its fluorescence polarization

(reflecting the ability to freely rotate in the lipid biolayers) strongly depends on the

microenvironment, with decreases in membrane microviscosity resulting in increased

fluorescence polarization. In this study, BODIPY-PC-labeled exosomes were sonicated in the

presence of AA-PEG-DSPE lipid, and fluorescence polarization was recorded (Fig. 2). Co-

incubation of PTX with exosomes in the absence of sonication did not alter membrane
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microviscosity; however, small but statistically significant fluidization of exosomal membranes

was recorded when a high amount of lipid (0.5 µg/ml) was added to the solution. Next,

significant decreases (more than two times) in membrane microviscosity were recorded upon

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sonication that is consistent with our previous observations (15). The fluidity of exosomal

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membranes was partially restored when PTX was added to the solution. Sonication of exoPTX in

the presence of the lipid further increased membrane microviscosity up to naïve non-sonicated

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exosomes (Fig. 2). Noteworthy, the greater amount of lipid was added to the solution; the higher

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the microviscosity levels obtained. We hypothesize that the sonication leads to dysregulation of

exosomal membranes and creation of additional space for PTX molecules. This resulted in an
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increased LC for PTX. The incorporation of high amounts of lipid molecules upon sonication

allowed sealing membrane bilayers that may impede PTX loading, and as a result, diminish LC
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(Fig. 1 A).
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3.3 Accumulation of AA-PEG-exoPTX in Target Cancer Cells in vitro

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The ability to deliver the drug payload into target cells is crucial for the therapeutic efficacy of

exosomal formulations. Although the molecular function of sigma receptors is not yet fully
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defined, there is increasing evidence that these receptors are overexpressed in many cancer cells

(24). First, we validated sigma receptor as a target for LLC murine model of pulmonary

metastases. Western blot analysis revealed high expression levels in murine LLC cells (3LL-

M27), as well as murine lung adenocarcinoma cells (344SQ), human small-cell lung carcinoma

cells (H69/AR), and human non-small cell lung carcinoma cells (A549), and low, if any, in

normal human lung fibroblasts (Hel 299) (Supplemental Fig. 1 A). The protein bands were

quantified and normalized to beta actin (Supplemental Fig. 1 B). This indicates that the choice

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of AA (a sigma receptor ligand) as a vector to target 3LL-M27 cells is appropriate for the

presented experiments in the murine LLC model, as well as for further clinical evaluations.

Previously, we demonstrated that accumulation levels of fluorescently-labeled exosomes in 3LL-

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M27 cells was considerably greater (about 30 times) then accumulation of liposomes or

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polystyrene nanoparticles (15). Herein, the receptor-mediated accumulation of DiL-labeled

vectorized exosomes (AA-PEG-exo) was studied in target 3LL-M27 cells in vitro, and compared

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against control non-vectorized sonicated exosomes (exo), as well as exosomes with incorporated

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PEG-DSPE lipid without AA targeting moiety (PEG-exo) (Fig. 3 A). The obtained data

indicated that vectorized AA-PEG-exo nanocarriers were taken up in much higher quantities than
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non-vectorized sonicated exosomes. The PEGylated exosomes without AA-targeting moiety

were taken up less than parental exosomes, probably due to the PEG chains blocking interaction
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of exosomal surface proteins, which assist in cell accumulation.

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To further assess the capability of incorporated AA to target exosomes to sigma receptor, a

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competitive inhibition study was carried out in 3LL-M27 cells (Fig. 3 B). In this experiment,

3LL-M27 cells were pre-treated with free AA at varying concentrations, washed with PBS, and
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then equal amounts of vectorized AA-PEG-exo along with free AA were added to the cells for

one hour. Fluorescence levels were measured; the amount of exosomes/μg protein was

quantified and graphed against the concentration of AA (Fig. 3 B). Results showed a dose-

dependent response of AA-PEG-exo to competitive inhibition by increasing concentrations of

free AA, indicating that AA-PEG-exo were taken up by receptor-mediated endocytosis.

Noteworthy, even a large amount of free AA added to the AA-vectorized exosomes was not able

completely inhibit exosome uptake in target cells, suggesting involvement of other exosomal

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surface proteins in this process, for example LFA1 (as demonstrated by western blot, Fig. 1 B)

that is consistent with our recent report (23).

The importance of exosomal surface proteins in assisting in exosome take-up was confirmed in

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accumulation studies using proteinase K treatment to strip exosomes of surface proteins (Fig. 4).

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In this experiment, DiL-labeled sonicated AA-vectorized (AA-PEG-exo) and non-vectorized

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sonicated exosomes (exo/sonic), as well as non-sonicated naïve exosomes (exo/naive) were

incubated with 3LL-M27 cancer cells for various times; and the number of taken exosomes was

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accessed by fluorescence. The obtained data indicate that accumulation levels in target cells
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increased in order: exo/naïve < exo/sonic < AA-PEG-exo (Fig. 4). This confirmed our previous

reports that treatment with ultrasound improved exosome accumulation in cancer cells (22), as
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well as neuronal PC12 cells (15). In parallel, the same exosomal formulations were treated with

proteinase K, and added to the cells. The digestion of the exosomal surface proteins significantly
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decreased uptake by target cells in all formulations. These results clearly show the advantages of
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exosome-based drug delivery systems over common synthetic nanocarriers related to the

facilitated uptake of exosome carriers by means of surface adhesive proteins. Noteworthy,

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stripping of surface proteins from vectorized exosomes (AA-PEG-exo) decreased their transport

at significantly lesser extent than non-vectorized exosomes (Fig. 4), probably due to the assisted

AA-mediated accumulation in cancer cells.

Finally, to assess the effect of AA-vectorization on intracellular trafficking of exosomes, 3LL-

M27 cells were supplemented with DiL-labeled AA-PEG-exo and exo as a control, and then

stained with MitoTracker, or LysoTracker, or ERTracker dyes to visualize mitochondria,

lysosomes, or endoplasmic reticulum (ER), respectively (Supplemental Fig. 2). Confocal

images revealed that exosomes preferentially distribute in order: lysosomes > ER >
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mitochondria. Noteworthy, the intracellular fate of exosomes was not altered by the addition of

a vector to the sigma receptor.

3.4 Co-localization of Systemically-administered Exosomes with Pulmonary Metastases in

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LLC mice

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To assess the ability of AA-vectorized exosomes to target pulmonary metastases, studies were

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conducted in the LLC mouse model with 3LL-M27 cells overexpressing the optical reporter

green fluorescent protein (GFP). In particular, we investigated the percentage of exosomes co-

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localized with cancer cells in the lungs. To induce metastases, C57BL/6 mice were injected with
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GFP/3LL-M27 (5x106 cells/100 μL) as described above. Twenty-one days later, DiL-labeled

autologous non-vectorized (exo) and AA-vectorized (AA-PEG-exo) exosomes were injected i.v.
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to C57BL/6 tumor-bearing mice. Four hours later, mice were sacrificed, perfused; lungs were

sectioned on microtome and examined by confocal microscopy. Confocal images revealed 94.4
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± 0.8% of AA-exosomes in immunohistochemistry slides were co-localized with lung metastases

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(Fig.5 D-F) indicating efficient targeting of AA-exoPTX in vivo. In contrast, only 21.8 ± 0.2% of

systemically-injected non-vectorized exosomes were co-localized with cancer metastases (Fig. 5

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A-C). Noteworthy, no AA-exosomes were found in the lungs of healthy animals (Fig. 5 G-J).

This suggest that systemically-administered AA-exosomes can efficiently reach pulmonary

metastases and deliver their drug payload to target cancer cells.

It was reported that along with pulmonary metastases, i.v. injected 3LL- M27 cells populate liver

in C57BL/6 mice (32). We investigated, whether AA-vectorized exosomes were able to target

liver metastases similar to pulmonary metastases. As expected, confocal images showed close to

complete co-localization of fluorescently-labeled AA-PEG-exosomes with cancer cells in the

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liver (Supplemental Figure 3) suggesting that AA-exosomal formulations may be suitable for

other than pulmonary metastases therapy.

3.5 Therapeutic Efficacy of AA-PEG-exoPTX Against Lung Metastases

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C57BL/6 mice with established mCherry-3LL-M27 (red) metastases were systemically injected

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with AA-PEG-exoPTX. Mice injected with exoPTX, or Taxol, or saline, or exosomes alone

(without PTX) were used in control groups. Eighteen days later mice were sacrificed, perfused,

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and lung slides were examined by confocal microscopy (Fig. 6 A). The images demonstrated a

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superior antineoplastic efficacy of AA-exoPTX compared to non-vectorized exoPTX, or Taxol

that resulted in the potent eradication of pulmonary metastases. Additional images of randomly
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selected lung slides are shown on Supplemental Figure 4. The quantitative assessment of

metastases area on the lung slides of animals is shown on Fig. 6 B. The survival studies
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confirmed these results (Fig. 6 C). Administration of AA-exoPTX formulation caused a

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significantly stronger suppression of metastases growth and greater survival time. Noteworthy,

injections of control empty (naïve) exosomes without PTX incorporated did not slow down
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tumor growth in mice with pulmonary metastases. This confirms the superior antineoplastic
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efficacy of AA-PEG-exoPTX upon systemic administration, as compared to Taxol, or non-

vectorized exoPTX formulation.

4. DISCUSSION

The efficient targeted delivery of anticancer agents to pulmonary metastases remains one of the

greatest challenges for the treatment of lung cancer. Several nanoformulations are being studied

in clinical trials, or have already been approved by the FDA for use in humans (33, 34).

However, conventional nanoparticles have limited biocompatibility, and normally are cleared

rapidly from the circulation by the MPS (4). We have developed a new drug delivery system that
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is based on natural vectors, exosomes, released by autologous macrophages for the delivery of

PTX. We utilize macrophage-derived exosomes that exert unique biological activity reflective of

their origin, and therefore, can provide advantages of both nanotechnology, and cell-mediated

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drug delivery that is based on the innate functions of immune cells.

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Using exosomes as drug delivery vehicles offers a number of benefits over common drug

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administration regimens; however, there are some limitations and challenges that need to be

addressed. One of the major challenges is the efficient loading of exosomes without significant

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alterations to the structure and content of exosomal membranes. PTX is a highly hydrophobic
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compound that is likely to be incorporated into the inner region of the relatively tight and highly

structured lipid bilayers of exosomes. Therefore, we developed a specific procedure wherein

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lipid bilayers are reshuffled upon mild sonication. To target exosomal carriers to cancer cells, we

incorporated a vector moiety with AA that is known to specifically bind to the sigma receptor
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using the same sonication procedure. Herein, we optimized the amount of incorporated AA
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vector moiety that did not impede with the loading of PTX. The obtained AA-PEG-exoPTX

formulation showed an extraordinary ability to accumulate in target cancer cells; these exosomes
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were taken up via receptor-mediated endocytosis in considerably greater numbers than non-

vectorized exosomes in vitro. Noteworthy, addition of AA moiety did not alter intracellular

trafficking of exosomal formulations in cancer cells.

The most interesting results were obtained in the mouse LLC model. Our data demonstrate a

robust accumulation and nearly complete co-localization of systemically administered AA-

vectorized exosomes with cancer metastases. We hypothesized that both AA-vector, and LFA1

protein expressed on exosomal membranes were responsible for this preferential accumulation in

pulmonary metastases upon systemic administration. Significantly, exosomes were targeted to

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liver metastases as well; a complete co-localization of AA-vectorized exosomes with cancer cells

was demonstrated in this work. Taking in to account that hydrophobic fluorescent dye DiL may

serve as a model drug that has a low solubility in water (for example PTX), it suggests that

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exosomes can target cancer metastases and deliver their cytotoxic payload specifically to these

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cells avoiding healthy tissues. The investigations of trafficking of exosomes and their

components (loaded therapeutic agents, as well as exosomal lipids, and proteins, etc.) are on the

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way in our laboratory.

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Finally, a systemic administration of AA-vectorized exosomal formulation of PTX resulted in
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the superior antineoplastic effect and increased survival times in mice with pulmonary

metastases, as compared to exoPTX or Taxol treated animals. We hypothesized that this effect
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was due to the superior therapeutic efficacy of the formulation. Specifically, the efficient

targeting of PTX incorporated into AA-vectorized exosomes resulted in the potent inhibition of
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pulmonary metastases growth. The confocal images and quantification of metastases area on
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lung slides confirmed this suggestion. Furthermore, targeting of PTX to the cancer cells may

also decrease side effects of the formulation that also will manifest in increased survival times.
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Notable, LLC cells are known to express the Pgp drug efflux transporter in vivo (35). We

demonstrated earlier that the incorporation of PTX into exosomes may not only increase its

solubility, but also allow for overcoming of Pgp-mediated drug efflux in resistant cancer cells.

This effect may be attributed to differences in internalization routes for exoPTX and Taxol.

Exosomes and micelles, such as those found in Taxol, enter resistant cancer cells by endocytosis,

however exosomes have a superior accumulation due to the presence of adhesion proteins,

tetraspanins, integrins, immunoglobulins, proteoglycans, and lectins (36), which are not found on

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artificial nanocarriers. Furthermore, exosomes consist of cellular membranes that may fuse with

the plasma and/or endocytic membranes and deliver their cargo, bypassing Pgp-mediated efflux.

Moreover, it is known that exosome-mediated cell-to-cell communication is key in the battle

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between cancer and the immune system (37). Thus, Parolini et al. (38) showed that exosome

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fusion with target cells occurs more efficiently under acidic conditions, implying that exosomes

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may be taken up preferentially by tumors (which have an acidic microenvironment) rather than

the surrounding healthy tissue. Finally, decoration of exosomes with PEG chains may

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considerably increase their circulation in the blood as was demonstrated earlier (28).
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Regarding the elimination fate and blood circulation time, the source of exosomes is of great

importance. Thus, when exosomes released by cancer cells, they efficiently accumulated in MPS
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and quickly eliminated from the blood stream (38, 39). Contrary, exosomes released by primary

fibroblast-like mesenchymal cells can bypass immune clearance by monocytes and macrophages
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that results in their prolonged blood circulation. The reported mechanism is related to the CD47
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molecule that expressed on the surface of exosomes and its binding partners thrombospondin-1

(TSP1) and signal regulatory protein α (SIRPα) that produce the “don’t eat me” effect in
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macrophages (40). This may also enhance specificity of exosomal formulations to cancer cells in

MPS-reach organs. Further studies of the mechanism of these superior antineoplastic effects are

ongoing in our lab.

Overall, all four mechanisms mentioned here are likely to have significant impact on AA-PEG-

exoPTX anticancer activity, i.e.: (i) vector-mediated preferential accumulation in cancer cells,

(ii) efficient delivery of incorporated cargo into target cancer cells, (iii) bypassing Pgp-mediated

drug efflux in resistant cancer cells, and (iv) prolonged circulation time in the blood via

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PEGylation. In conclusion, exosomes promise an unparalleled efficacy in the treatment of many

life-threatening conditions, including those lacking effective pharmacotherapy.

5. ACKNOWLEDGMENTS

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We are grateful to Dr. Leaf Huang for the invaluable comments and suggestions.

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FUGURE LEGENDS

Figure 1. Characterization of exoPTX and AA-PEG-exoPTX formulations. PTX and vector

moiety AA-PEG-DSPE was incorporated in exosomes by sonication procedure. A: Loading

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Capacity (LC) for PTX in vectorized exosomes was measured by HPLC. Increasing amounts of

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vector moiety resulted in LC decrease. The highest concentration of AA-PEG-DSPE that did not

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lower the LC for PTX in exosomes was chosen for subsequent experiments (shown by arrow). B:

Western blot data indicated that formulations retained the exosome markers TSG101, flotillin,

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and LFA1, specific marker for lymphocytes, after the sonication procedure. C: Size was
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measured by NTA and DLS, the zeta potential was measured by DLS. The data was obtained

from five independent experiments.

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Figure 2. Effect of AA-PEG-DSPE incorporation and PTX loading on fluidity of exosomal

membranes. BODIPY-PC-labeled exosomes were examined by fluorescence polarization

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measurements. The ultrasound treatment significantly decreased microviscosity of exosomal

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membranes compared to naïve exosomes. The microviscosity of sonicated exosomes was

increased with PTX loading and then further increased with incorporation of the lipid upon
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sonication. Values are means ± SEM (n = 4). Symbols indicate the relative level of significance

compared with naïve exosomes (p < 0.05). The data was obtained from three independent

experiments. Symbols indicate the relative level of significance compared to protein-striped

exosomes (*p < 0.05, **p < 0.005).

Figure 3. Accumulation of AA-vectorized exosomes in cancer cells. A: 3LL-M27 cells were

incubated with fluorescently-labeled AA-PEG-exo, or PEG-exo, or exo for various times, and

accumulation levels were measured. AA-PEG-exo were more readily taken up by 3LL-M27 as

compared to exo and PEG-exo. B: AA-PEG-exo formulation showed a dose-dependent response

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to competitive inhibition by AA, indicating that this formulation entered cells by receptor

mediated endocytosis. Results are expressed as number of exosomes/μg protein vs. concentration

of AA. The data was obtained from three independent experiments.

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Figure 4. Importance of surface proteins on exosome accumulation in 3LL-M27 cancer

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cells. Naïve (circle), sonicated (triangle), and AA-vectorized (cross) exosomes were incubated

with Proteinase K to strip surface proteins and subsequently labeled with DiL; accumulation of

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Proteinase K-treated exosomes (dashed line) or control exosomes (solid line) was examined in

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cancer cells. Stripping the exosomal surface proteins resulted in significant decreases in exosome

accumulation levels in target cells for all formulations. The data was obtained from three
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independent experiments.

Figure 5. Co-localization of systemically-delivered AA-exosomes with pulmonary

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metastases. Exosomes were isolated from macrophages conditioned media and labelled with
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DiL dye (red, A, D). C57BL/6 mice were i.v. injected with 3LL-M27 cells transduced with

lentiviral vectors encoding the optical reporter GFP fluorescent protein (green, B, E). 7 days
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later, tumot-bearing mice were i.v. injected with DiL-labeled non-vectorized exosomes (red, A),
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or AA-exosomes (red, D). Four hours later, mice were euthanized, perfused, lungs were

sectioned, and stained with DAPI (blue). Confocal images revealed significant co-localization of

AA-exosomes with metastases (94.4 + 0.8 %, F) that was greater than those of non-vectorized

exosomes (21.8 + 0.2 %, C). No exosomes were found in lungs of healthy animals without

metastases (G-J). Bar: 20 µm. The data was obtained from three independent experiments.

Figure 6. AA-PEG-ExoPTX induced potent anticancer effect against lung metastases in

LLC mouse model. A: C57BL/6 mice with established Luc/mCherry-3LL-M27 lung metastases

were i.v. treated with: saline, or Taxol, or exoPTX, or AA-PEG-exoPTX, or exosomes alone

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(no drug). 18 days later, mice were sacrificed, perfused, and lungs slides were examined by

confocal microscopy. AA-PEG-exoPTX treatment resulted in a potent inhibition of metastases

(red) that was more effective than treatment with Taxol or exoPTX. The bar: 50 μm. B:

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Quantitative assessment of metastases area on the lung slides of animals treated with: 1) saline,

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or 2) Taxol, or 3) exoPTX, or 4) AA-PEG-exoPTX, or 5) exosomes alone. **p < 0.005. C: A

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survival of C57BL/6 mice with established metastases was recorded for four treatment groups:

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1) saline (dimonds), or Taxol (squares), or 3) exoPTX (triangles), or 4) AA-PEG-exoPTX

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(crosses), or 5) exosomes alone (stars). A superior effect on suppression of metastases growth

and greater mice survival was recorded in AA-PEG-exoPTX treatment group (n = 6). The data
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was obtained from three independent experiments.
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Graphical Abstract
Exosomes released by autologous macrophages were loaded with paclitaxel (PTX) and vectorized
with anisamide-polyethylene glycol (AA-PEG) moiety to target the sigma receptor, which is
overexpressed by lung cancer cells. The obtained formulation (AA-exoPTX) showed a high loading
capacity, profound ability to accumulate in cancer cells upon systemic administration, and

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improved therapeutic outcomes.

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