Ultrasound Facilitates Naturally Equipped Exosomes Derived from

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Biological and Medical Applications of Materials and Interfaces

Ultrasound Facilitates Naturally Equipped Exosomes Derived from Macrophages and Blood Serum for Orthotopic Glioma Treatment Lianmei Bai, Yichen Liu, Kaili Guo, Kun Zhang, Quanhong Liu, Pan Wang, and Xiaobing Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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ACS Applied Materials & Interfaces

Title: Ultrasound Facilitates Naturally Equipped Exosomes Derived from Macrophages and Blood Serum for Orthotopic Glioma Treatment Authors: Lianmei Bai#, Yichen Liu#, Kaili Guo, Kun Zhang, Quanhong Liu, Pan Wang*, Xiaobing Wang*

Affiliation: National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710119, China

*Corresponding author: Pan Wang & Xiaobing Wang

#Author Contribution: The authors contributed equally to this work.

E-mail: [email protected]; [email protected]

Tel: +86-29-85310275

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ABSTRACT: Exosomes (Exos) are endogenous nanocarriers that have utility as novel delivery systems for the treatment of brain cancers. However, in general, natural Exos show limited BBB-crossing capacity and lack specific targeting. Further modifications including targeting peptides and genetic engineering approaches can circumvent these issues, but the process is time-consuming. Focused ultrasound (FUS) has been approved by the FDA for the diagnosis and treatment of brain disease due to its non-invasive nature, reversibility, and instantaneous local-opening of the BBB. In this study, we developed a natural and safe transportation system using FUS to increase the targeted delivery of Exos for glioma therapy. We also compared the advantages of macrophage derived Exos (R-Exos) and blood serum derived Exos (B-Exos) to screen for an improved platform with scope for clinical transformation. In vitro, both R-Exos and B-Exos were transported through BBB models and accumulated in glioma cells with the assistance of ultrasound exposure. R-Exos and B-Exos displayed no obvious differences in physical characteristics, drug release, tumor targeting, and cytotoxicity when combined with FUS. In vivo, animal imaging studies suggested that the fluorescence intensity of B-Exos plus single FUS in tumors were 4.45-fold higher than B-Exos alone. Furthermore, B-Exos plus twice FUS


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treatment, efficiently suppressed glioma growth with no obvious side effects. We therefore demonstrate that the combination of FUS and naturally abundant B-Exos are a potent strategy for brain cancer therapeutics. KEYWORDS: Exosomes, Blood-brain barrier, Drug delivery, Focused ultrasound, Glioma theranostics 1. INTRODUCTION Glioma is one of the most lethal brain tumors worldwide.1 Due to invasive growth, the complete surgical removal of gliomas is almost impossible. To date, surgical methods have combined radiotherapy and chemotherapy which can alleviate symptoms and prolong the survival of glioma patients. However, these procedures are accompanied by serious trauma and a loss of quality of life. 2,3 The blood-brain barrier (BBB) that exists between the central nervous system (CNS) and peripheral blood circulation prevents many anti-tumor drugs from reaching the brain, severely hindering the efficiency of glioma therapeutics.4,5 Diverse organic and inorganic nano-formulations have therefore been developed to overcome the BBB.6,7 Whilst drug-loaded nanoparticles in the bloodstream can be confronted with systemic toxicity and rapid clearance by the mononuclear phagocyte system (MPS), they greatly improve trans-BBB delivery efficacy. In recent decades, endogenous nanocarrier-exosomes (Exos) as drug delivery systems have received widespread attention due to their low toxicity, low immunogenicity and excellent biocompatibility.8 Exos are a class of membrane



secreted lipid vesicles consisting of a lipid bilayer and aqueous core (40-150 nm) that can enclose complex molecules including proteins, nucleic acids, lipids and sugars, to form an endogenous transport system permitting cellular exchange.9,10 Pioneering studies have shown the utility of gene/drug delivery using Exos to cross the BBB following systemic administration,8, 11 providing new opportunities for CNS disease treatment.12 Exos of diverse origin have been explored and distinct advantages and disadvantages are reported.13 Macrophage derived Exos (named R-Exos) bear MHC II on their surface and can present antigens directly to T-cells, playing an immune-protective role.14 Recent studies have revealed that macrophages derived Exos facilitate drug transfer to cells of the neurovascular units including neurons, astrocytes, and brain microvessel endothelial cells, implying therapeutic potential for brain neurological disease.15 In addition to cell-derived Exos, blood serum-derived Exos (named B-Exos) are more widely available, are of higher yield, and have some extent of targeting capability.16 It is reported the blood serum-derived Exos are from a variety of cells, mainly including reticulocytes, white blood cells and blood platelets, etc.17 Exos derived from different tumor cells, inflammatory cells, healthy cells, and blood serum are being studied for various applications. However, a paucity of studies have focused on the distinction between Exos of different origins, their unique biological activity, and ability to cross the BBB. Moreover, although some Exos possess an intrinsic ability to cross biological barriers, the delivery of therapeutic drugs to entire tumors at concentrations that permit effective treatments, remain largely undefined.


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To improve BBB permeation, studies have focused on chemical modifications and genetic engineering, but these studies are time-consuming, labor-intensive, and success is not guaranteed. Focused ultrasound (FUS) is a proven non-invasive strategy that can produce transient, reversible, and local BBB disruption.18 Chen et al. reported that FUS can open the BBB and improve the delivery of IL-12 to brain tumors.19 In addition, FUS can enhance the targeted delivery of drug particles in the lesion through focusing and positioning of the exposed area. Several drug carriers including microbubbles, liposomes, and micelles combined with FUS have been developed as promising strategies to increase local drug concentrations, reducing systemic side effects.6, 20 Based on this evidence, we investigated the utility of FUS to assist BBB opening and to promote Exos mediated enrichment at the tumor site in situ. Herein, we demonstrate the design of a drug delivery system that combines FUS with doxorubicin (Dox) loaded Exos derived from macrophages and blood serum, for glioma diagnostics and therapy (Scheme 1). Using FUS, Exos without surface-alterations were assessed and their natural therapeutic potential, safety, and feasibility were investigated. In addition, we evaluated the unique biological activity of Exos based on their origin, which may influence their ability to cross the BBB. We compared basic parameters including natural targeting, US-assisted cellular uptake, BBB crossing, and toxicity to glioma cells between macrophage- and blood serum-derived Exos. Finally, the anti-tumor effects elicited by FUS and Dox-loaded Exos were evaluated on orthotopic gliomas. The combined drug delivery system displayed unique advantages including: (i) non-invasive BBB opening by



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extracorporeal FUS; (ii) biocompatibility and safety by autologous nanovesicles; (iii) a combination of FUS and Exos, particularly B-Exos of sufficient abundance that can be used for the delivery of anticancer drugs or genes in clinic practice.

Scheme 1. Schematic illustration of the administration. First, R-Exos-Dox and B-Exos-Dox were prepared by a mild and green method. The first FUS (1 MHz, 1 W, 1 min) was employed to open BBB to promote the targeting enrichment of Exos in the tumor site; then, a second exposure was applied to stimulate local drug release and exert site-specific therapeutic action.

2. MATERIALS AND METHODS 2.1. Chemicals. Doxorubicin hydrochloride and ethidium bromide (EB) were purchased

from

Sigma-Aldrich

(St.

Louis,

MO,

USA).

3-(4,

5-Dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT), Hoechst 33258, the fluorescent dyes 3, 30-dioctadecyloxacarbocyanine perchlorate


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(DIO) and 1, 1 0-dioctadecyl-3, 3, 30, 30-tetramethylindotricarbocyanine iodide (DIR) were purchased Invitrogen (Thermo Scientific Inc., USA). Dulbecco’s modified Eagle’s medium (DMEM) medium, penicillin-streptomycin and fetal bovine serum (FBS) were acquired from Gibco Life Technologies (AG, USA). All other reagents were commercial products of analytical grade. 2.2. Cell culture and animals. The luciferase-transfected mouse glioblastoma cell line GL261 were kindly provided by Prof. Chengren Li (Army Medical University of Chinese PLA), and the mouse brain endothelial cells bEnd.3, mouse colon carcinom CT26 cells, C6 glioma cells, mouse embryo fibroblasts NIH/3T3 cells and murine macrophage RAW264.7 cells were obtained from the Cell Resource Center of Chinese Academy of Science. All cells lines were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1mM L-glutamine, and cultured in an incubator with 5% CO2 and 100% humidity at 37°C. The C57BL/6 mice (18-20 g) were supplied by the Experimental Animal Center of Fourth Military Medical University (FMMU) (Xi’an, China) and housed at room temperature with a 12 h light/dark cycle and allowed free access to food and water. The animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shaanxi Normal University (Xi’an, Shaanxi, China). For the establishment of gliomas in situ, mice were anaesthetized by injection with 5% chloral hydrate, then affixed in stereotactic head frame. The hair of the mice head was removed and made a vertical incision on the skull skin, then



marked the anterior fontanelle and the striatum (striatum: 0.6 mm posterior and 1.8 mm lateral from the bregma point). GL261-luc cells (1 × 106 in 5 µL DMEM) were intracranial implanted to the right hemisphere of the brain at a depth of 3 mm from the surface over 5 minutes using a microsyringe. Holding the needle for 5 min, the needle was removed slowly, and bone wax was used to close the hole, and sutured the mice skin incision finally. Tumors can be detected by bioluminescence at 7 days after implantation. 2.3. Ultrasound apparatus. The experimental ultrasound (US) devices were the same as that described in previous studies.21 For in vitro cell experiments, a cell-based therapeutic pulsed ultrasound apparatus (Sheng Xiang High Technology Co. Ltd., China) was applied, and such ultrasound parameters (1.0 MHz, 0.6 W/cm2, duty cycle of 20 %, duration of 1 min) were used. The ultrasound exposure system used in vivo experiments consisted of a power amplifier (AG1020, T&C power conversion Inc., USA), and a focused transducer with frequency of 1.0 MHz and a focal length about 5 cm (ndtXducer, Northborough, MA). 2.4. Exos isolation. RAW264.7 cells were incubated in DMEM media containing 10% Exos-depleted fetal bovine serum. Exos-depleted bovine serum was obtained by ultracentrifugation at 120000×g for 18 h at 4°C. When cells reaching a confluency of 60–80% in culture flasks, the culture media was collected and centrifuged at 3000×g for 15 min and 10000×g for 45 min to remove cells and cell debris. The collected supernatant was filtered through a 0.22-μm filter to remove impurity. After filtering, the specially-made Exos isolation reagent based on previous studies with slight


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modifications was added to the filtrate at ratio of 1:4 (w/w).22 The medium and reagents were thoroughly mixed until a homogeneous solution is obtained. The samples were incubated overnight at 4°C, and then centrifuged at 6500×g for 25 min at 4°C. Blood samples were collected from the orbit venous plexus of C57BL/6 mice to acquire fresh serum. Fresh serum was centrifuged at 3000×g for 10 min followed by 12,000×g for 30 min at 4°C. The collected supernatant was passed through 0.22 μm filter to remove impurity. Then transferred filtrate to a fresh conical tube, and added the specially-made Exos isolation reagent at a ratio 1:4 (w/w). Samples were mixed thoroughly by inversion, and incubated at 4°C overnight. The samples were centrifuged at 6500×g for 25 min at 4°C to pellet Exos. Removed the supernatant and re-suspended Exos in PBS for immediate use or storage at -80°C for later experiment. Protein concentration of Exos was estimated by BCA assay. 2.5. Characterization of Exos. Exos were diluted in filtered PBS and maintained at 4°C. The average vesicle diameter and zeta potential were obtained by NTA ZetaView®. The morphology of the Exos was characterized by transmission electron microscopy (TEM). Briefly, Exos were adsorbed onto carbon-coated copper grids for 30 min at room temperature and a small drop of phosphotungstic acid was added to the copper grid for 15 min and washed three-times with water. The grid was air-dried overnight and imaged using a Hitachi H-600 TEM at an accelerating voltage of 80 KV.



2.6. Western blot. The Exos were detected by western blot as described.23 Briefly, Exos derived from RAW264.7 cells and blood serum were re-suspended into RIPA lysis buffer containing 1 mM EDTA for protein extraction. Lysates were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose membrane (Whatman International Ltd, UK). Membranes were rinsed with PBS for several minutes and blocked with Odyssey blocking buffer for 1 h. The membranes were incubated with primary antibodies against CD63, CD81 and ALIX (1:1000 dilution, Abcam), followed by incubation with secondary antibodies. Images were acquired with an Odyssey infrared imaging system and analyzed using software specified by the Odyssey systems. 2.7. Drug loading and release. Exos derived from RAW264.7 macrophages and blood serum were diluted in PBS to a concentration 1 μg/μL total protein. For high-drug loading efficiency, a series of ratios were assessed. Dox and Exos were thoroughly mixed and incubated at 37°C for 2 h at specific ratios. Exos isolation reagent was added to the Exos and mixed with Dox at a ratio of 1:4 w/w for 4 h at 4°C. Exosomal Dox (Exos-Dox) were obtained by centrifugation and washing in PBS. The encapsulation percentage of Dox was determined through spectrophotometric methods. The Exos-Dox bilayer was disrupted with 1% Triton X-100 to release encapsulated drug. The concentration of Dox in the solution was determined using an UV−vis spectrophotometer at 490 nm. The drug loading (DL) and encapsulation efficiency (EE) were then calculated. The release of Dox from Exos-Dox was performed in physiological conditions ± FUS treatment, which was monitored as


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previously described with minor modifications.24 Drug release profiles were constructed to evaluate the influence of FUS on drug release. Briefly, 1 mL of Exos-Dox solution with or without FUS was transferred into a dialysis bag and added to 50 mL of buffer. At different time points, 200 µL of buffer was removed for UV-Vis spectrophotometer analysis and an equal volume of fresh buffer was added to the dialysis tubing. The cumulative release of the Dox fraction was calculated based on the standard curve of the corresponding buffer solution. 2.8. Cellular uptake assays. GL261 cells were seeded at a density of 2 × 105 cells/mL on 35-mm dishes and allowed to adhere overnight. R-Exos-Dox and B-Exos-Dox were added to GL261 cells for 12 h at 37°C. Cells were collected, resuspended in serum-free DMEM, and analyzed by flow cytometry. Identical methods were used to analyze the uptake of Exos by CT26, NIH/3T3, U87, C6, and GL261 cells, respectively. Confocal microscopy was used to assess the intracellular trafficking of Exos. Briefly, GL261 cells were grown on glass bottom dishes at a density of 2 × 105 cells/mL, and treated with free Dox, DIO labeled Exos, and DIO labeled Exos-Dox with or without US, respectively. After 12 h, cells were washed three times in PBS, fixed in paraformaldehyde for 15 min and stained with Hoechst 33258 prior to imaging. The accumulation of fluorescently-labeled Exos was visualized by confocal microscopy. 2.9. Cell viability assay. GL261 cells were seeded at 2 × 105 cells/mL on 35-mm



dishes and allowed to attach for 12 h. Different doses of Dox in free or exosomal forms were added to the Opti-MEM for 2 h. Then, dishes were irradiated with the indicated ultrasound treatment. The viability was determined at 24 h after different treatment with MTT assay . 2.10. Blood circulation of exosomal Dox. C57BL/6 mice were randomly grouped (n=3 per group) and intravenously injected with Dox, R-Exos-Dox and B-Exos-Dox at a Dox dose of 5 mg/kg. Approximately 200 µL of blood samples from each group were collected into heparinized tubes at 30 min and 1, 2, 3, 4, 8, 12 and 24 h post-administration. Samples were centrifuged at 5000 rpm for 10 min to harvest the plasma. Dox was methanol extracted and samples were centrifuged at 10000 rpm for 10 min. The Dox concentration in the supernatants was examined basing on standard curves measured on a microplate reader. 2.11. In vitro BBB model. Endothelial cell barrier passage and the US permeabilized transportation of the Exos was assessed in a transwell system. Briefly, 1 × 104 cells were seeded into the upper wells with a pore size of 8 μm. DMEM was added to the lower chamber. After incubation for 5 days, free DIR and Exos-DIR were added to cells in the upper wells in the presence or absence of ultrasound. MBs were added to the culture medium of the sonication groups. The fluorescence intensity of DIR in the lower chamber was assessed on a microplate reader. To further investigate if US can increase the targeted accumulation of Exos in glioma cells, an in-vitro BBB model was established according to previous reports.25


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Briefly, 1 × 104 cells were seeded into the upper inserts and GL261 cells were plated onto the lower chambers of the transwell at a density of 2 × 103 cells/compartment. Cells were then incubated for 5 days. For the sonication group, MBs were diluted in culture medium and sonicated for 60 s with a 10 ms pulse length. Following US treatment, drugs were added to the apical compartment of the BBB model. The final concentration of Dox was 10 µg/mL. After 24 h, the fluorescence intensity of GL261 cells in the basolateral compartment was determined by fluorescence microscopy and flow cytometry. 2.12. FUS accelerates the BBB-crossing potency of Exos in vivo. To evaluate the capacity of Exos to cross the intact BBB and to assess whether FUS can promote this process, normal mouse models with an intact BBB were randomly assigned into three groups. The first group was injected intravenously with Exos-DIR solution (5 mg/mL, 200 µL per mice). The second group was injected with Exos-DIR solution 2 h post-sonication. The control group was injected with DIR alone. Whole-animal imaging

was

recorded

using

an

IVIS

spectrum

imaging

system

12

h

post-administration (IVIS 100, USA). In mice treated by heart perfusion, brains were removed and placed in a dish. Ex vivo fluorescence imaging and average intensities were recorded using an IVIS system. EB permeation in the brains of each treatment group were also analyzed. We further explored the tumor accumulation of Exos assisted by FUS. C57BL/6 mice bearing orthotopic GL261 tumors were randomly assigned into the three groups. Images were captured at 0, 2, 4, 8, 12, 24, 48 and 72 h after injection. At 12 h, mice



were sacrificed and ex vivo imaging of the excised organs was performed. Imaging data were processed and analyzed on IVIS Living Image 3.0 software. 2.13. In vivo anti-glioma efficacy. To evaluate antitumor efficacy, GL261-bearing mice were randomly divided into five groups. The FUS (1MHz, 1 W, 1 min) group had the BBB temporally disrupted and 5 mg/kg of free Dox or Exo-Dox were injected into the caudal vein at 2 h. A second FUS was performed at 12 h to trigger drug release and site-specific therapeutic effects (twice FUS). This procedure was followed every 3 days on 3 occasions. Bioluminescence imaging (BLI) was performed using the Xenogen IVIS Lumina II system (Perkin Elmer, Waltham, MA, USA). Approximately 8 min post-intraperitoneal injection of D-luciferin, animals were imaged and the procedure was repeated. Signals in the region of interest were quantified in units of mean photons, per second, per square cm, per steradian. 2.14. Histopathological evaluation. For histological analysis, tumors and major organs were fixed in 10% formalin for 24 h, and washed twice with PBS. Paraffin embedded tissue sections were stained with H&E and observed through microscopy. 2.15. Statistical analysis. All data are presented as mean ± standard deviation (S.D.). Significant differences between the groups were performed using a t-test. A p-value less than 0.05 was considered to be of significance for all comparisons. 3. RESULTS AND DISCUSSION 3.1. Preparation and characterization of Exos-Dox. The size and zeta potential of unmodified R-Exos and B-Exos were determined by NTA techniques (Particle Metrix,


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Germany). The average diameter of R-Exos was ~120 ± 35 nm whilst B-Exos was around 90 ± 25 nm (Figure 1A). The loading of Dox with R-Exos and B-Exos was estimated through the vesicle dimensions, which increased to 130 ± 30 and 100 ± 34 nm in R-Exos-Dox and B-Exos-Dox, respectively. The vesicles had a regular size and shape when assessed by TEM (Figure 1B). The size stability of the particles showed no obvious aggregation in PBS at 4°C during the 7 day period (Figure 1C), indicating good storage stability. In addition, the time-dependent stability of vesicles in PBS, DMEM and PBS containing 20%, or 50% FBS at 37 °C showed no significant changes in particle size, and were steady by 24 h, suggesting in vivo biocompatibility (Figure 1D). Such stable features were accompanied by their small size could facilitate blood circulation and the accumulation of vesicles in cancers through the enhanced permeability and retention (EPR) effect. For the zeta potential (Figure 1E), both R-Exos and B-Exos showed negative values, that increased after Dox loading (R-Exos-Dox and B-Exos-Dox increased from -16.6 ± 0.36 to -12.2 ± 0.42 and -13.4 ± 0.46 to -10.3 ± 0.26, respectively). Dox is positively charged, meaning its loading increases the potential of distinct carriers.26 These results also indirectly supported Dox loading into Exos. We also detected CD63, CD81 and Alix on R-Exos and B-Exos, indicating that both vesicle preparations contain CD63, and that a tetraspanin typically enriched the surface of the Exos (Figure 1F).



Figure 1. Characterization of Exos derived from macrophages and blood serum with or without Dox-loading. (A) Size distribution of R-Exos, R-Exos-Dox, B-Exos, and B-Exos-Dox measured by nanoparticle tracking analysis (NTA). (B) TEM images of R-Exos, R-Exos-Dox, B-Exos, and B-Exos-Dox. Scale bars: 100 nm. (C) NTA analysis of diameter-distributions of R-Exos, R-Exos-Dox, B-Exos, and B-Exos-Dox after different storage times at 4°C. (D) Stability of vesicles in PBS, DMEM and 20%, or 50% FBS at 37 °C. (E) Zeta potential of vesicles analyzed by NTA. (F) Western blot analysis of exosomal marker proteins on R-Exos and B-Exos (20 μg of total protein). Error bars represent the S.D. for n = 3.

3.2. Drug loading and FUS boosted drug release from Exos. To date, therapeutic agents have been encapsulated into Exos using various passive and active methods,


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including incubation at room temperature (RT) with or without saponin permeabilization, electroporation, freeze-thaw cycles, sonication, extrusion and dialysis.27 These approaches result in variable loading efficiencies and stabilities of the drugs in Exos. For Dox, electroporation and magnetic separation provide higher drug loading into Exos,

28

however, the loading efficiency is not the only factor that

must be considered. Exos membrane integrity and stability are important for drug delivery. In this system, we optimized the drug-loading capacity of vesicles using a mild and efficient system that caused no surface modifications from co-incubation at RT with different weight ratios. The encapsulation efficiency (EE %) and drug loading efficiency (DL %) of Exos-Dox prepared using different mass ratios were calculated using standard curves of Dox concentrations at a fluorescence intensity of λex = 490 nm, λem = 580 nm (Figure S1). As shown in Table S1, S2, when the mass ratio of Dox and R-Exos was 1:15, the DL % of doxorubicin was 4.9%, and the EE % was 74%. For B-Exos, the maximum DL % was 4.6% when the ratio was 1:20, and the EE % was up to 96.6%. Gao et al. obtained Exos-Dox through incubation, and the Dox encapsulation efficiency and loading capacity were 9.06% and 2.60%, respectively.29 Compared to other methods, although the efficiency of passive incubation at room temperature was low, the structure and stability of the Exos remained high, owing to the incubation at RT being a gentle method that led to no structural damage. The Exos from both sources were heterogeneous in size, composition, and biogenesis. Thus, the drug loading efficiency may be dependent on both the source and enrichment of proteins on the surface.30



The fluorescence (FL) spectra of Dox, R-Exos-Dox, and B-Exos-Dox are shown in Figure 2A. The fluorescence spectra of Dox, R-Exos-Dox and B-Exos-Dox showed comparable emission spectra, however, the FL intensity of R-Exos-Dox and B-Exos-Dox was much lower than that of free Dox, suggesting that sufficient loading of Dox by Exos could induce its FL self-quenching. Moreover, the absorption spectra of Dox, R-Exos-Dox, and B-Exos-Dox were tested (Figure 2B). Compared to free Dox, the spectra of R-Exos-Dox and B-Exos-Dox showed no changes in the position or shape of absorbance bands, while the peak values were somewhat lower, which may be due to the compression of Dox in the lumen of Exos and resulting in insufficient photon absorption.

Figure 2. (A) Fluorescence spectra and absorption spectra (B) of Dox, R-Exos-Dox and B-Exos-Dox at Dox concentration of 30 µg/mL. (C) Release profiles of Dox from B-Exos and R-Exos in PBS (pH 7.4) without shake at 4°C; (D) Release profiles of Dox from R-Exos with different FUS intensity in PBS (pH 7.4) with shake at 37°C; (E) Release profiles of Dox from B-Exos with different FUS intensity in PBS (pH 7.4) with shake at 37 °C. (F) The


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pharmacokinetics of Exos-Dox in vivo. Error bars represent the S.D. for n = 3.

Exos represent versatile and advanced nano-delivery systems for the loading of anticancer drugs including doxorubicin, curcumin and paclitaxel, and other gene targeting systems including siRNA and miRNA. These endogenous vesicle-based systems can improve drug solubility and pharmacokinetics, ultimately increasing treatment efficacy without compromising safety.31 In addition to drug loading, the release of endogenous nanoparticles is critical to their pharmacological efficiency. Recent work has focused on the combination of drug delivery vehicles with ultrasound-mediated drug release. Husseini et al. explored the effects of cavitation on Dox release from folate-conjugated micelles.32 To evaluate the drug-release behavior of exosomes in a static status or flowing conditions when combined with FUS, we first assessed the in vitro release profiles of Dox from R-Exos and B-Exos at pH 7.4 at 4°C without shaking. Figure 2C shows that little drug release occurred at 48 h in both Exos preparations (26.94% for R-Exos and 30.06% for B-Exos). However, under pH 5.0, both R-Exos-Dox and B-Exos-Dox exhibited a relatively rapid and massive release behavior (68.6% and 70.65% at 48 h), since the decrease of pH would result in Dox protonation and accelerate its release (Figure S2A). This suggests that Dox could be released from exosomes after entering the acidic environment of late endosomes and lysosomes of cancer cells. After 48 h, the drug release of R-Exos-Dox and B-Exos-Dox still remained around 40 % in either 20% or 50% FBS, indicating that the exosomes could protect Dox in bloodstream (Figure S2B). We next assessed the impact of FUS on the release of Dox at pH 7.4 with shaking at 37°C, thus simulating



an in vivo environment. As shown in Figure 2D-E, the vesicles released the drug (R-Exos: 26.4% and B-Exos: 28.4% in 48 h) in the absence of FUS irradiation, indicating that the Exos exhibit relatively slow release behavior. For R-Exos, drug release was approximately 45.77%, 52.33%, and 64.32% at 12 h at different intensities of FUS irradiation for 1 min when LP was 1W, 2W and 3 W respectively. For B-Exos, drug release was comparable to R-Exos, which was approximately 52.73%, 58.32%, and 70.20% at 12 h with 1W, 2W, 3W FUS treatment, respectively, which was followed by a sustained and slow release over the next 48 h. Moreover, increasing the FUS intensity resulted in accelerated Dox release, suggesting that FUS initiates cargo release from Exos. A number of FUS-triggered release mechanisms for drug-loaded Exos are plausible. The multiple effects of FUS, including its mechanical shearing action and cavitation effect, play important roles in vesicle structure instability.33 For tumor killing, targeted drug release at the lesion site is preferable. When the drugs reach the lesion site and accumulate to high levels, the applied FUS promotes a burst of drug-release, leading to more efficient tumor killing. We therefore speculate that Exos combined with FUS is an excellent drug delivery system that promotes targeted accumulation and controlled drug release. 3.3. Exosomal Dox displays an increased blood circulation time. The influence of exosomal encapsulation on the pharmacokinetics of Dox were determined through the measurement of serum drug concentrations (Figure 2F). The Dox in R-Exos and B-Exos exhibited similarly high concentrations initially, and being gradually cleared from the circulation over time. There is no significant difference in the elimination


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half-life (t1/2) between R-Exos-Dox and B-Exos-Dox. The AUC (0–∞) area of R-Exos and B-Exos were 1.67- and 1.46-fold higher than that of free Dox, respectively. At 24 h, the concentrations of R-Exos-Dox and B-Exos-Dox in the blood were 2.26 and 2.70-fold higher than free Dox, indicating that the Exos significantly prolong the circulation of Dox in the blood, conducive to drug accumulation at the tumor site.34 From previous studies it is known that the incorporation of cholesterol and sphingomyelin (SM) into liposomes decreases the rate of clearance by RES.35 Whilst Exos derived from different sources contains elevated cholesterol and SM,36 the unique lipid composition of Exos can also influence their in vivo circulation. 3.4. Cellular uptake and cytotoxicity. Therapeutic drugs exert their anti-tumor activity based on the intracellularly accumulated doses. Confocal laser scanning microscopy was used to track the intracellular localization of Exos and Dox. We used DIO to track exosomal membranes (Figure 3A,3B), and after incubation with GL261 cells for 12 h, green DIO signals were dispersed within the cytoplasm, suggesting that both R-Exos and B-Exos were taken up by the cells, and that the red fluorescence of Dox dissociated from Exos and redistributed into the nucleus consistent with previous studies. We observed no significant change in the distribution of macrophage derivedand blood serum derived- Exos in tumor cells. Exosomal Dox displayed higher levels of intracellular uptake than free Dox, which was further enhanced by US treatment. The uptake of Exos has been linked to a range of phagocytosis and endocytic mechanisms, with endocytosis the most common route. Micropinocytosis involves non-specific sampling of the extracellular environment.37,


38

The mechanism(s) of


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cellular uptake of the drugs under US treatment is more complex.39 Recent studies suggest that US treatment enhances drug uptake in part as a result of sheer stress and the formation of transient membrane pores. Pores formed upon US treatment are rapidly resealed, suggesting that other processes contribute to the enhanced uptake.40,41 Recent studies suggest that US enhances clathrin-mediated endocytosis (CME) and fluid-phase uptake through distinct mechanisms, which may improve targeted drug delivery. 42, 43

Figure 3. Cellular uptake of Exos. (A) Intracellular distribution of R-Exos and Dox at 12 h incubation with GL261 cells. (B) Intracellular distribution of B-Exos and Dox at 12 h incubation with GL261 cells. (C) Macrophage cells uptake of R-Exos-Dox and B-Exos-Dox at 12 h. (D) GL261 cells uptake of R-Exos-Dox and B-Exos-Dox at 12 h. (E) GL216 tumor cells uptake of R-Exos-Dox and B-Exos-Dox after different incubation time. (F) Cellular uptake of R-Exos-Dox and B-Exos-Dox in CT26, NIH/3T3, U87, C6 and GL261 cells at 12 h.

##p

cells, **p < 0.01 versus NIH/3T3 cells. Error bars represent the S.D. for n = 3.


< 0.01 versus CT26

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To evaluate the cellular uptake performance of vesicles, intracellular Dox content was determined by flow cytometry. R-Exos-Dox and B-Exos-Dox were incubated with GL261 cells and RAW264.7 cells, respectively. For RAW264.7 cells, the uptake efficiency of Exos and self-derived Exos were similar to those derived from blood serum at 12 h. (Figure 3C). For GL261 cells, R-Exos-Dox and B-Exos-Dox did not greatly differ (Figure 3D). Next, we analyzed the cellular uptake efficiency of R-Exos and B-Exos by GL261 tumor cells at different time points. The uptake efficiency of R-Exos were 18%, 57.3%, 78.2% and 84.3% for incubation times of 3, 6, 9 and 12 h, respectively. For B-Exos, the cellular uptake efficiencies were 10.2%, 20.4%, 50.2% and 79.5% for 3, 6, 9, and 12 h, respectively. These results imply that Exos are internalized into cells in a time-dependent manner (Figure 3E). Furthermore, we compared the uptake efficiency of R-Exos and B-Exos in different cell types. As shown in Figure 3F, the uptake efficiency of tumor cells is higher than that of normal cells under identical conditions. As previously described, multiple internalization mechanisms regulate the uptake of Exos into cancer cells. Phagocytosis and endocytosis are often receptor-mediated to ensure specificity. The transferrin receptor, endothelin B receptor, survivin, and integrin’s are overexpressed on multiple cancer cells which may facilitate Exos internalization.44 As determined by MTT assays, neither R-Exos nor B-Exos exhibited cytotoxicity when incubated with GL261 cells for 24 h, even at the highest concentration of 100 µg /mL, indicating that both Exos as drug carriers are relatively safe (Figure 4A and 4B). We then compared the toxicity of the different treatments on the viability of



GL261 cells. Briefly, cells were incubated with free Dox (0, 0.01, 0.1, 1, and 10 µg/mL) and exosomal Dox with or without US. As shown in Figure 4C and 4D, the cytotoxicity of Dox, R-Exos-Dox and B-Exos-Dox were concentration-dependent (Figure S3). At the same dose, the cytotoxicity of Exos-Dox was higher than that of free Dox. The loss in viability due to the Exos-Dox was approximately 46.67% at concentration of 10 µg/mL and the presence of US decreased viability to 26.57% (p < 0.01). This result is likely caused by US treatment eliciting enhanced cellular uptake and drug-release kinetics from Exos-Dox. Using this method, the two sources of Exos displayed comparable cytotoxicity.

Figure 4. Cytotoxicity of Exos-Dox on cultured GL261 cells. The cytotoxicity of Blank-Exos derived from (A) RAW264.7 cells and (B) blood serum. Cell proliferation after combination treatment of (C) R- Exos-Dox plus US and (D) B-Exos-Dox plus US (n = 3; *p < 0.05, **p < 0.01). Error bars represent the S.D. for n = 3.

3.5. FUS promotes the BBB permeability of Exos in vitro. BBB is a major


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physiological barrier that prevents drugs or drug delivery systems from reaching the brain. Researchers have developed distinct strategies to overcome or bypass the BBB, including penetrating through BBB by cellular internalization, temporarily opening BBB and intranasal delivery.45, 46 The ability of Exos across the BBB in vitro and in vivo models was assessed in our study.47, 48 Previous studies have shown that US can open the BBB,

49

so an appropriate US intensity was selected as an auxiliary means

for BBB accessibility. The US equipment is shown in Figure 5A in which cell-based in vitro trans-well models of the BBB were validated. Figure 5B shows the co-culture of the in vitro BBB model of bEnd.3/GL261 cells. BBB permeability was assessed on a microplate reader and fluorescence microscopy. DIR and Exos-DIR were added to the upper chamber and the time-dependent transmigration to the lower chamber was determined through the relative light absorption of each sample. As shown in Figure 5C and 5D, at 0-1.5 h after US, the BBB permeability gradually increased, reaching its maximum at 2 h, and remaining stable thereafter. The free DIR barely crossed the BBB model. However, DIR delivered by Exos from RAW264.7 cells or blood serum were able to cross the intact BBB, the levels of which were enhanced by US. Similarly, as shown in Figure 5E and 5F, the fluorescence observation demonstrate that the Dox content in the bottom chamber of GL261 cells was significantly increased after Exos combined with US, being consistent with the above results.



Figure 5. Ultrasound promoted Exos mediated delivery across the intact BBB. (A) Schematic diagram of the ultrasound equipment in vitro. (B) Illustration of the in vitro BBB model. (C) Absorbance of DIR and R-Exos-DIR in the lower chamber with or without US treatment at different time points. BBB permeability of Exos was tested by Microplate reader. Experiments were run in duplicates. Mean and standard deviation are reported. (D) Fluorescence of DIR and B-Exos- DIR in the lower chamber with or without US treatment at different time points. (E) Fluorescence microscopy observation of R-Exos and B-Exos uptaken by tumor cells after crossing the BBB with or without US treatment, scale bar = 200 µm. (n = 3; *p < 0.05, **p < 0.01). Error bars represent the S.D. for n = 3. (F) Quantitative analysis of (E).

3.6. FUS facilitates exosomal drug delivery in orthotopic glioma. The US equipment is shown in Figure 6A. Evans blue extravasation was used to evaluate the BBB-permeability of B-Exos. Due to the restriction of time- and culture resources-consuming, Exos derived from cells were far from meeting the needs of


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many in vivo experiments and possible clinical applications. In order to acquire abundant Exos, Exos derived from blood were taken into consideration, because of the abundant source, high-yield, easy access and natural brain targeting ability. We initially evaluated the capacity of B-Exos to cross the intact BBB using normal mouse models. B-Exos-DIR was employed as a tracer of exosomes (by using the NIR fluorescence of DIR) to assess the nanoparticles’ accumulation in brain tumors, using IVIS spectrum imaging system. As shown in Figure 6B, B-Exos penetrated the brain by transportation across the intact BBB, and a significant fluorescent signal was detected in FUS treated groups. However, no accumulated fluorescence was detected in the brains of mice administered the same dose of DIR alone. Fluorescence quantification shows that the intensity of Exos-DIR + FUS was 4.45-fold higher that Exos-DIR without FUS (Figure S4). Together, these results clearly demonstrate that Exos can transfer drugs across the BBB into the brain and that FUS further promotes this process.



Figure 6. Ultrasound-mediated B-Exos crosses the BBB and in vivo targeting distribution in tumor-bearing mice. (A) Schematic diagram of the ultrasound equipment in vivo. Evans Blue extravasation is shown (left: without FUS; right: with FUS, 1 MHz, 1 W, 1 min). (B) Above: in healthy mice NIR fluorescence imaging at 12 h post DIR-loaded Exos injection (From left to right: control, Exos, Exos+FUS); Below: ex vivo fluorescence images of isolated brains at 12 h after intravenous administration of DIR-labeled blood Exos. (C) In GL261 orthotopic mice NIR fluorescence imaging at different time points post DIR-loaded Exos injection. (Upper: without FUS; lower: with FUS, 1 MHz, 1 W, 1 min). (D) Ex vivo NIRF imaging of the main organs of GL261 orthotopic mice at 12 h after intravenous administration of DIR-labeled B-Exos. (E) Quantitative analysis of the NIRF signals of (C) (**p < 0.01 versus group). (F) Quantitative analysis of the NIRF signals of (D), **p < 0.01 versus group.

The clinical outcomes of glioma using chemotherapeutics are insufficient due to the existence of the BBB and the non-targeted delivery of drugs. The targeting distribution of drug-loaded nanocarriers in tumors could potentially enhance the antitumor activity of these drugs in vivo. Various nano-formulations have therefore been developed to achieve the brain tumor targeting of macromolecular therapeutics. Further, using DIR labelled B-Exos, we tracked the kinetic accumulation of B-Exos in GL261-bearing orthotopic gliomas. As shown in Figure 6C, E, in the absence of FUS treatment, the FL signal of Exos selectivity accumulated in the brain at 4 h and reached a maximum at 12 h post- injection, suggesting passive EPR-based tumor targeting of Exos. Through comparison, FUS treatment led to significant accumulation of the FL signal in the tumor site at 2 h which peaked at 12 h after intravenous injection. Moreover, each excised organ was examined 12 h post-injection (Figure 6D, F). Similarly, ex vivo images of the isolated brain demonstrated that DIR-labeled blood Exos following FUS treatment accumulated to


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higher levels in the brain compared to without FUS treatment, indicating that selective irradiation by FUS promotes drug accumulation in the tumor site. By the statistical analysis of NIR images at 12 h post-injection, without FUS treatment, B-Exos mainly accumulated into the liver, followed by the spleen, kidneys, brain and lungs. This is in agreement with previous studies.50 FUS trigger did not show much influence on the distribution of B-Exos in other organs, and B-Exos also displayed high level in liver, then followed by brain, spleen, and kidney. It should be note that, the distribution of exosomes in different organs could be influenced by metabolic pathways after injection into the body, and the enrichment in each organ is changing with time.51 Our results show that exosomes could accumulate into the brains, while focused ultrasound further greatly enhanced the accumulation and distribution in the targeting exposed gliomas. Previous studies have shown that the interaction between transferrin and transferrin receptor (TfR) can facilitate the brain targeting of blood Exos24, 41, which may also contribute to the above processes. 3.7. FUS enhanced Exos chemotherapy in orthotopic gliomas. The ability of FUS to effectively overcome BBB and the selective glioma targeting properties of Exos nanocarriers offers therapeutic opportunities for the treatment of brain cancer. To test this hypothesis, we used Exos-Dox combined FUS to evaluate the anti-tumor activity. Seven days post implantation, mice were imaged on an Xenogen IVIS Lumina II system to monitor tumor development, and treatment protocols were initiated as described in Figure 7A. Briefly, nanoparticles were injected 7 days post-tumor cell implantation with a Dox dose of 5 mg/kg, with 3 repeats on 3 days intervals. The



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antitumor efficacy was investigated through single or twice FUS treatment. The first FUS was performed 1 h prior to B-Exos-Dox administration, and the second FUS was introduced

12

h

post

B-Exos-Dox

injection.

Orthotopic

luciferase-tagged

GL261-bearing mice were divided into five groups: (a) saline as a control (n=6), (b) Dox + twice FUS, (c) Exos-Dox, (d) Exos-Dox + single FUS, (e) Exos-Dox + twice FUS. On days 0, 5, 10 and 15 after treatment, BLI was used to monitor tumor growth (Figure 7B). The therapeutic responses indicated by BLI signals were quantified and the result is shown in Figure 7C. On day 15 of treatment, both Dox + twice FUS and Exos-Dox groups exhibited modest inhibitory effects on tumor growth. Exos-Dox treatment showed a delayed loss of body weight and longer survival times compared to the Dox + twice FUS, which may be due to myocardial endothelial cells limiting Exos-Dox crossing, and avoiding the accumulation of drugs in the heart, resulting in lower side effects. The Exos-Dox plus single FUS group inhibited tumor growth to a greater extent than Exos-Dox, which could be attributed to FUS exposure promoting the enrichment of drugs at the tumor site through the opening of BBB. Importantly, the combination of Exos-Dox with twice FUS treatment resulted in a visible regression of tumor growth and an extended survival time (Figure 7E), leading to a significant improvement over free Dox and Exos-Dox treatment. After twice FUS treatments, the tumor suppressive effects were improved in comparison to single FUS treatment, which could be explained by the fact that the second FUS triggered efficient Dox release from Exos-Dox. In summary, Exos combined with twice FUS stimuli in specific sequences achieves an excellent therapeutic effect in orthotopic gliomas.


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H&E staining of the glioma and TUNEL immunohistological assays were used to evaluate the in vivo antitumor effects (Figure 7D). H&E staining revealed obvious tumor tissue damage caused by Exo-Dox plus twice FUS. TUNEL staining showed almost no apoptotic signal in the saline group, and some apoptotic cells in the Dox plus twice FUS and Exo-Dox groups. Exo-Dox plus twice FUS treatment resulted in higher rates of apoptosis compared to the other groups.

Figure 7. Exos encapsulated Dox combined FUS effectively improved anti-glioma activity.　(A) Diagram illustrating experimental procedure. (B) In vivo BLI images of GBM orthotopic tumor bearing mice that were treated with different protocol, i.e., saline, Dox + twice FUS, Exos-Dox,



Exo-Dox plus single FUS and Exo-Dox plus twice FUS. (C) Quantitative analysis (n = 5) of the BLI signals of (B). (D) Microscopic observation of tumor sections by H&E staining (upper) and TUNEL reaction (lower); scale bar = 100 µm. (E) Animal survival curves in different groups.

3.8. Evaluation of side effects. The toxicity of drug formulations was correlated to their chemical structure, surface charge, in vivo bio-distribution, metabolism and clearance. Despite the therapeutic efficacy of Exo-Dox plus FUS, potential adverse effects should be examined. Amongst the side effects, cardio toxicity is a major problem caused by anthracyclines. H&E analysis of the heart, liver, spleen, and kidney was performed (shown in Figure 8A). The hearts of Dox plus twice FUS treated mice showed vacuoles and moderate myofibril disorganization. In Exos-Dox -treated mice with or without FUS, the hearts were similar to the control group, and it was speculated that myocardial endothelial cells limit Exo-Dox crossing, avoiding accumulation of the drug in the heart.52 No other major organs exhibited any significant pathological changes. Simultaneously, the body weight of tumor-bearing mice was measured during the experiment. As predicted, the body weights of the saline group decreased during the period and no major changes occurred in response to other treatments (Figure 8B). In addition, the biochemical detection of GOT and GPT levels suggested no acute hepatic damage caused by the designed treatment protocol (Figure S5). Together, these data provide a relatively safe evaluation of our platform.


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Figure 8. Evaluation of side-effects. (A) H&E staining showing no obvious histopathological changes of major organs post distinct treatment (scale bar = 50 μm). (B) Effect of different treatments on mouse body weight (mean ± S.D., n = 5).

4. CONCLUSION In this study, we report experiments that compare and analyze the production and therapeutic effects of Exos derived from different sources loaded with Dox. We show that both R-Exos and B-Exos could effectively deliver Dox into glioma tumors with the help of FUS, whilst with no significant difference in physical features, ultrasound-responsiveness, and specific glioma targeting were observed. Due to the abundant source, high-yield, and easy access of blood serum derived Exos, they hold higher clinical potential than cellular sources. The platform using a combination of FUS and natural Exos provides a valuable future strategy for glioma and other brain diseases. ASSOCIATED CONTENT Supporting Information Supporting Information is shown as supplementary material.



AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (81571834), the Innovative Talents Promotion Plan in Shaanxi Province (2017KJXX-78), and the Academic Leaders and Academic Backbones, Shaanxi Normal University (16QNGG012), and the Fundamental Research Funds for the Central Universities (GK201802002, 2016TS056). REFERENCES (1) Zhang, Y.; Zhang, L.; Hu, Y.; Jiang, K.; Li, Z.; Lin, Y. Z.; Wei, G; Lu, W. Cell-permeable NF-KappaB Inhibitor-Conjugated Liposomes for Treatment of Glioma. J. Controlled Release. 2018, 289, 102-113

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Ultrasound Facilitates Naturally Equipped Exosomes Derived from

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