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Blood Exosomes Endowed with Magnetic and Targeting Properties for Cancer Therapy Hongzhao Qi, Chaoyong Liu, Lixia Long, Yu Ren, Shanshan Zhang, Xiaodan Chang, Xiaomin Qian, Huanhuan Jia, Jin Zhao, Jinjin Sun, Xin Hou, Xu-bo Yuan, and Chunsheng Kang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06939 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Blood Exosomes Endowed with Magnetic and Targeting Properties for Cancer Therapy Hongzhao Qi,†,┬ Chaoyong Liu, ┴,┬ Lixia Long, † Yu Ren,‡ Shanshan Zhang,† Xiaodan Chang,† Xiaomin Qian,† Huanhuan Jia, ‡ Jin Zhao, † Jinjin Sun,§ Xin Hou,†,* Xubo Yuan,†,* Chunsheng Kang┴,* †Tianjin Key Laboratory of Composite and Functional Materials, School of Material Science and Engineering, Tianjin University, Tianjin 300072, China, ‡Tianjin Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China, §Department of Surgery, Second Hospital of Tianjin Medical University, Tianjin 300211, China, ┴Laboratory of NeuroOncology, Tianjin Neurological Institute, Department of Neurosurgery, Tianjin Medical University General Hospital and Key Laboratory of Neurotrauma, Variation, and Regeneration, Ministry of Education and Tianjin Municipal Government, Tianjin 300052, China. ┬These authors contributed equally to this work. KEYWORDS: exosome, superparamagnetic nanoparticle clusters, scalable separation, drug delivery, cancer targeting

Abstract: Exosomes are a class of naturally occurring nanoparticles that are secreted endogenously by mammalian cells. Clinical applications for exosomes remain a challenge

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because of their unsuitable donors, low scalability, and insufficient targeting ability. In this study, we developed a dual-functional exosome-based superparamagnetic nanoparticle cluster as a targeted drug delivery vehicle for cancer therapy. The resulting exosome-based drug delivery vehicle exhibits superparamagnetic behavior at room temperature, with a stronger response to an external magnetic field than individual superparamagnetic nanoparticles. These properties enable exosomes to be separated from the blood and can target diseased cells. In vivo studies using murine hepatoma 22 subcutaneous cancer cells showed that drug-loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth. Our developments overcome major barriers to the utility of exosomes for cancer application.

Targeted drug delivery has proven to be a critical issue in achieving efficient cancer therapy.1 In recent decades, endogenous vehicles, such as protein and polysaccharide nanoparticles, have been explored extensively for drug delivery to obtain better therapeutic outcomes because of their low toxicity and biocompatibility.2-5 Exosomes are a class of naturally occurring nanoparticles that are secreted endogenously by mammal cells, and they have been recognized recently as excellent vehicles for drug and gene delivery.6,7 They are spherical 30-100 nm vesicles,8 which makes them small enough to passively diffuse into tumors via the enhanced permeability and retention (EPR) effect. Also, these nanoparticles have excellent in vivo stability and less cytotoxicity over synthetic vehicles.9 Attempts have been made to develop exosomebased vesicles for the delivery of various therapeutic cargos, ranging from small molecule chemo-therapeutics and anti-inflammatory agents to miRNA and siRNA.10-13 Despite advances in exosome-based drug delivery, many challenges still exist. For example, the development of a scalable protocol to achieve large-scale exosome production for clinical applications is a problem


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that has not been addressed to date. In terms of safety and secretion levels, a human cell-type that is ideal for exosome derivation must be identified. Furthermore, endowing exosomes with cancer-targeting ability remains a technical challenge. Exosomes are secreted by many cell types and are present in in vitro culture media and in in vivo body fluids.14 Various studies have shown that exosomes can be separated from in vitro cell culture media, especially immune or tumor cells culture media.15 However, these cells secrete too few exosomes (~6-12 µg per 106 dendritic cells cultured for 7 d)13 and may provide immuneand cancer-stimulating activities because of their protein and nucleic acids cargoes from donor cells.16,17 Compared with in vitro sources, blood is a better source of exosomes. As a major source of exosomes in the blood stream, reticulocytes (RTCs) release 1014 (at least 200 µg) exosomes per day during their maturation into erythrocytes.18 RTC-derived exosomes contain various membrane proteins, including transferrin (Tf) receptors, but are devoid of any immunestimulating activity and cancer-stimulating properties and thus are a potential source of sufficient and safe exosomes.18, 19 However, no reports exist on drug delivery by RTC-derived exosomes. The development of RTC-derived exosomes as drug carriers needs to achieve simultaneous efficient blood-separation and targeted delivery. The separation protocol should be technically simple for operation, should not impair the structural integrity (at least the size) of exosomes and should facilitate drug-loading and clinical administration. Exosomes should possess a targeting ability to reduce the side effects of loaded chemo-therapeutics that result from the nonspeciﬁc distribution of exosomes in organs.10 Protocols that have been reported for exosome separation and targeting functionalization have not been tailored for RTC-derived exosomes. Traditional ultracentrifugation for exosome separation is laborious, and the resulting exosome pellet, which is usually contaminated with proteins, cannot be re-dispersed, which results in difficulty in


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intravenous injection.20 Although antibody affinity magnetic beads (usually hundreds of nanometers or several microns) have been used extensively to separate exosomes from body fluids, it is difficult to disperse the resulting exosome-bearing magnetic particles in solution because of their ferromagnetic characteristics.21 Recently, a novel purification method has been reported in which iron oxide magnetic nanoparticles have been incubated with donor cells.22 These cells could secrete exosomes loaded internally with magnetic nanoparticles. Exosomes can be separated magnetically and manipulated by magnetic force for targeting and subsequent delivery of drug payload against cancer cells. However, this technique is performed in cell culture and cannot be transferred to RTC-derived exosomes. Similarly, incorporation of targeting peptides or proteins into exosomes by inducing their expression in exosome donor cells is also not feasible for RTC-derived exosomes.13 Developing a new delivery platform that can realize simultaneous RTC-derived exosome scalable separation and targeted delivery is a key driver for exosome-based drug delivery. Superparamagnetic magnetite colloidal nanocrystal clusters (SMCNCs) show promise for biomedical applications. Cluster formation occurs by self-assembly or solution growth and results in a significant increase in nanocrystal magnetization, while retaining its superparamagnetic characteristics.23 Effective separation and purification of SMCNC-based drug carriers from solutions or their movement in blood can be controlled by using moderate magnetic fields (MFs). By using the concept of SMCNC, we have conceived a RTC exosome-based superparamagnetic nanoparticle cluster (denoted as SMNC-EXO) strategy for tumor-targeting drug delivery. In this strategy, multiple superparamagnetic nanoparticles (SPMNs) anchor onto each exosome to form a cluster, which increases the magnetization in a controllable manner


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while retaining its superparamagnetic characteristics. Such a strategy could separate exosomes from blood efficiently and provide exosomes with a robust targeting ability.

Scheme 1. Schematic illustration of construction and delivery of drug-loaded SMNC-EXOs: (I) collection of fresh serum from healthy mice, (II) incubation of M-Tfs with pre-dialyzed serum to form SMNC-EXOs, (III) magnetic separation and purification of SMNC-EXOs to remove blood proteins, (IV) re-dispersion of SMNC-EXOs, (V) DOX loading via incubation with SMNCEXOs, (VI) intravenous injection of DOX-loaded SMNC-EXOs into tumor-bearing mice.

RESULTS AND DISCUSSION Scheme 1 summarizes the cluster synthesis, with details given in subsequent sections. SMNC-EXOs were obtained directly from pre-dialyzed serum via magnetic separation, where


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the serum was extracted from the fresh blood of healthy mice (Step I). Incubation of SPMN-Tf conjugations (denoted as M-Tfs) with pre-dialyzed serum allowed multiple M-Tfs to bind to each RTC-derived exosome through a Tf-Tf receptor interaction to form SMNC-EXOs (Step II). Such unique structures increased the separation efficiency of exosomes while retaining a favorable dispersibility in solution once the external MF was removed. After being purified via magnetic separation and being re-dispersed into phosphate buffered saline (PBS) buffer, SMNCEXOs were obtained (Steps III and IV). Then, SMNC-EXOs with a phospholipid bilayer interacted with doxorubicin (DOX) through a hydrophobic effect to form drug-loaded SMNCEXOs (denoted as D-SMNC-EXOs) (Step V). After being injected intravenously into homologous mice bearing a hepatoma 22 (H22) cancer, D-SMNC-EXOs accumulated in the tumor region and released DOX to suppress tumor growth when an external MF was applied (Step VI). SMNC-EXO construction and characterization. To construct SMNC-EXOs, M-Tfs were first synthesized by conjugation of holo-transferrins (iron-loaded transferrin, holo-Tfs) to ~10 nm Fe3O4 SPMNs (Figures S1, S2, Supporting information). Procedures used for the construction and purification of SMNC-EXOs from pre-dialyzed serum are shown in Figure 1A. Briefly, 0.1 mg M-Tfs (containing ~8 µg Tfs) was mixed with 1 mL pre-dialyzed fresh serum and incubated for 4 h at 4°C to allow the M-Tfs to anchor onto exosomes via Tf-Tf receptor interaction. SMNC-EXOs containing ~20 µg exosome (exosome concentration was measured based on protein concentration)13 were obtained from the mixture by magnetic separation using a MF (1 T). The yield of exosomes increased with the increase in the amount of M-Tfs, but the largest yield obtained from 1 mL serum was ~105 µg. The yield was significantly greater than the traditional method from in vitro cell cultures (an average yield of 0.78 µg ± 0.14 µg exosome/mL


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of conditioned medium).24 Because of the formation of magnetic clusters when M-Tfs were anchored onto exosomes, the resulting SMNC-EXOs exhibit an enhanced response to the external MF compared with the M-Tfs. This was confirmed by the fact that the separation of SMNC-EXOs was significantly faster than that of M-Tfs (Figure S3). Upon removal of the external MF, the resulting purified SMNC-EXOs re-dispersed immediately and showed a high water-solubility in PBS, which is a typical superparamagnetic behavior. In contrast, exosomes separated using commercial magnetic beads (Life Technologies, Exosome-Human CD63 Isolation/Detection Reagent, USA) formed large complexes (Figure S4) and precipitated rapidly after re-dispersion (Figure S5), which demonstrates the unique advantage of the SMCNC-based separation method.


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Figure 1. A) Images of SMNC-EXOs construction process, B) representative TEM image of SMNC-EXOs, with insets of an enlarged detailed view and electron diffraction pattern, C) Western blot analysis of specific exosome marker proteins (CD9 and CD63) and Tf receptors in re-dispersed SMNC-EXOs, D) magnetic retention of M-Tfs and SMNC-EXOs in simulated blood circulation system (flow velocities: artery, 32.85 cm/s; vein, 14.60 cm/s; and capillary, 0.05 cm/s), red arrows are separated SMNC-EXOs and black arrow is separated M-Tfs.


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A transmission electron microscope (TEM, JEM-2100F, JEOL Ltd., Japan) image of SMNC-EXOs (Figure 1B) shows dark spots that surround spherical vesicles, which represent the cluster structures formed during synthesis. TEM energy spectrum analysis of the dark spots indicates the existence of elemental Fe (Figure S6); further electron diffraction revealed their Fe3O4 pattern, which indicates that the dark spots are SPMNs. Two typical exosome marker proteins (CD9 and CD63) and Tf receptors were detected by Western blot assay, which indicates that the vesicles are RTC-derived exosomes (Figure 1C).25 Figure S7 shows the size distribution of SMNC-EXOs measured by dynamic light scattering (BI-90Plus, Brookhaven Instruments Ltd., USA). Their hydrodynamic diameter (40 to 110 nm) was consistent with the size of typical exosomes as reported previously and indicates favorable dispersity of SMNC-EXOs in a PBS buffer.26 To prove SMNC-EXOs formed through Tf-Tf receptor interaction, we first incubated SPMNs with serum in the absence of Tfs, but no SMNC-EXOs were formed (Figure S8). This demonstrates that the formation of SMNC-EXOs relies on Tfs. In addition, since Tf-Tf receptor binding is sensitive to pH, we dispersed SMNC-EXOs in a pH 5.0 buffer to allow holo-Tfs to release iron and become apo-Tfs.27 Then the buffer pH was adjusted to 7.4. We found that the amount of separated exosomes decreased significantly (Figure S9) because of the dissociation of apo-Tfs with Tf receptors. This demonstrates that the SMNC-EXOs were formed through Tf-Tf receptor interaction. The RTC-exosomes could be separated directly from serum without pre-dialysis, but the separation requires a large amount of M-Tfs. Because the concentration of Kunming mice serum Tfs reaches ~0.8 mg/mL (~0.24 mg/mL holo-Tfs), at least 2.5 mg/mL M-Tfs (containing holo-


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Tfs ~0.2 mg/mL) is required to construct exosome-based SMNCs in serum, and the separation efficiency is low. Pre-dialysis of serum increases the separation efficiency significantly. SMNC-EXO stability. SMNC-EXOs stabilities were examined by monitoring changes in size with time by dynamic light scattering. SMNC-EXOs, with an average size of 80 nm, showed no obvious aggregation in PBS buffer at 4°C after 7 d, which indicates excellent storage stability in the solution (Figure S10A). In fresh serum at 37°C after 7 d, the magnetically separated and re-dispersed SMNC-EXOs also showed no obvious aggregation, which indicates their excellent application stability (Figure S10B). Although serum Tfs may dissociate M-Tfs reversibly from SMNC-EXOs, there is no significant change in the quantity of magnetically separated SMNCEXOs (Figure S11). This may be because SPMNs could shield M-Tfs, and to some degree, avoid the competition of serum Tfs. Even if a few M-Tfs competed for by serum Tfs became dissociated from SMNC-EXOs, the separation would not be influenced as long as the SMNC structures were maintained. Such stability accompanied by their small size could facilitate blood circulation and accumulation of SMNC-EXOs in cancers by the EPR effect. Magnetic-targeting ability of SMNC-EXOs. To evaluate the magnetic-targeting ability of SMNC-EXOs, a microfluidic system that controlled the SMNC-EXOs solution flow rates was used to simulate their retention under a moderate MF in blood circulation (Figure S12). Flow rates were held at 32.85 cm/s (artery), 14.60 cm/s (vein), and 0.05 cm/s (capillary).28-30 After injection, SMNC-EXOs were retained at the MF site under simulated vein and capillary blood flow rates (Video S1, Supporting information). This is consistent with previous reports that magnetic targeting is likely to be more effective in regions of slower blood velocity.31 Few MTfs were retained, even at the capillary blood flow rate, because of their low magnetization (Figure 1D). Cancer capillaries can occupy up to 50% of the total cancer volume in small cancers


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because of angiogenesis or neovascularization, and the mean blood flow rate in cancers may be considerably lower than in normal tissue perfusion.32, 33 Thus, following systemic administration, SMNC-EXOs could be retained and accumulate by magnetic interaction with an externally applied MF, thereby increasing the drug concentration in cancer lesions. Drug loading and release. We evaluated the drug-loading ability of SMNC-EXOs. A fluorescence spectra measurement was performed after incubation of SMNC-EXOs with Nile Red to probe exosome phospholipids for drug-loading. The Nile Red fluorescence intensity increased as the SMNC-EXO concentration increased from 0 to 452 µg/mL in aqueous medium (Figure 2A). The Nile Red fluorescence intensity of the same concentration of BSA solution is significant lower than that of SMNC-EXO solution (Figure S13). This suggests that SMNCEXOs possess a hydrophobic domain. Ultraviolet–visible absorption spectra proved that SMNCEXOs can increase the solubility of DOX (Figure S14) and the quantitative analysis indicated a DOX loading capacity of 10.25% (Figure S15). So, we can separate ~105 µg exosomes from 1 mL serum and load ~11 µg DOX in these exosomes. In addition, DOX-loading had little influence on SMNC-EXO size (Figure S16). To evaluate the drug-release behavior of D-SMNC-EXOs, we determined the in vitro release profile of DOX from SMNC-EXOs at pH 7.4 (physiological environment) and pH 5.0 (late endosome and lysosome). D-SMNC-EXOs released a small amount of drug at pH 7.4 (50% in 48 h), which indicates that SMNC-EXOs well protected the drug in blood circulation. At pH 5.0, D-SMNC-EXOs exhibited a relatively rapid and massive release behavior (80% in 8 h), followed by a sustained and slow release over a prolonged period of two days (Figure 2B). Decreasing the pH results in DOX protonation and accelerates DOX release, which suggests that


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DOX was released rapidly and massively from SMNC-EXOs after entering an acidic environment of late endosomes and lysosomes of cancer cells.

Figure 2. A) Fluorescence spectra of Nile Red after incubation with different concentrations of SMNC-EXOs, B) release profiles of DOX from D-SMNC-EXOs in PBS (pH 7.4) and acetate buffer (pH 5.0), C) intracellular distribution of SMNC-EXOs and DOX at 24 h incubation, right


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is the image observed along the cut plane, D) cellular uptake of D-SMNC-EXOs-mediated DOX by H22 cells as assessed by flow cytometry, DOX signal detection, E) cell viability of H22 cells exposed to different concentrations of DOX and D-SMNC-EXOs without external MF. Cytotoxicity, blood compatibility, and histocompatibility. A Cell Counting Kit-8 (CCK8) cell viability assay was performed on H22 cells to assess the cytotoxic effects of SMNCEXOs. In a 640 µg/mL SMNC-EXO solution, cells maintained as high as 90% viability (Figure S17). To study the biocompatibility of SMNC-EXOs in vivo, hemolytic activity tests and histological section analysis of major organs were performed to evaluate blood compatibility and histocompatibility, respectively. Neither hemolysis nor signs of acute organ injury were observed (Figures S18 and S19), which demonstrates that SMNC-EXOs are biocompatible as a drug delivery vehicle. Tumor cell uptake and inhibition. D-SMNC-EXOs labeled with fluorescein isothiocyanate (FITC), SMNC-EXOs labeled with FITC and free DOX were incubated with H22 cells for 24 h. Confocal laser scanning microscopy was used to track intracellular localization of SMNC-EXOs and DOX. As shown in Figure 2C, strong SMNC-EXOs signals (green) were dispersed within the cytoplasm, which suggests that D-SMNC-EXOs were taken up by the cells. The image observed along the cut plane indicates DOX dissociated from D-SMNC-EXOs distributed in the cytoplasm and nucleus at 24 h. To estimate cellular uptake efficiency, D-SMNC-EXOs labeled with FITC were incubated with H22 cells for 4 h. The cellular uptake efficiency of SMNC-EXOs without external MF was 63.76%, 81.44%, and 91.53% when the concentration of D-SMNC-EXOs was 50, 100, and 200 µg/mL, respectively (Figure S20), which implies that SMNC-EXOs were internalized into cells


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in a concentration-dependent manner. Exerting MF, however, reduced the cellular uptake efficiency of SMNC-EXOs (Figure S21A), probably due to the suspension characteristics of H22 cells. Because, in contrast with H22 cells, MF enhanced the adhesive-cultured Michigan Cancer Foundation-7 (MCF-7) cells uptake of SMNC-EXOs (Figure S21B). In line with the cellular uptake of SMNC-EXOs, the cellular uptake efficiency of DOX mediated by D-SMNC-EXOs increased significantly from 24.68% to 86.74% when the concentration of D-SMNC-EXOs increased from 60 µg/mL to 150 µg/mL (DOX concentration increased from 6 µg/mL to 15 µg/mL). Whereas, when the concentration of D-SMNC-EXOs reached 200 µg/mL (DOX concentration is 20 µg/mL), the cellular uptake efficiency increased only slightly to 91.44% (Figure 2D), which demonstrates that the cellular uptake of D-SMNCEXOs-mediated DOX is saturable. The growth-inhibition assay for H22 cells was tested. Cells were incubated with free DOX (0, 0.25, 0.5, 1, 2, 4, 8, 10 and 12 µg/mL) and D-SMNC-EXOs (with equivalent DOX) without external MF. As is shown in Figure 2E, the half maximal inhibitory concentrations (IC50) of DOX and D-SMNC-EXOs (without external MF) were 0.28 µg/mL and 0.37 µg/mL, respectively. Exertion of MF decreased the H22 suppression effect of D-SMNC-EXOs (Figure S22A) because of the low uptake efficiency of D-SMNC-EXOs under MF. In accordance with the results of uptake efficiency, MF enhanced the MCF-7 cell suppression effect of D-SMNCEXOs (Figure S22B). This suggests that SMNC-EXOs can enhance the suppression efficiency of DOX under external MF when D-SMNC-EXOs make sufficient contact tumor cells in vivo. In vivo distribution of SMNC-EXOs. The cancer-targeting ability of D-SMNC-EXOs was investigated using Kunming mice bearing a subcutaneous H22 cancer as a model. We labeled 1


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mg/mL D-SMNC-EXO with fluorescence probe Cy5.5, which was then injected intravenously into tumor-bearing mice. After 24 h administration, the in vivo biodistribution of Cy5.5-labeled D-SMNC-EXOs was monitored using a non-invasive near infrared fluorescence (NIRF) optical imaging technique by setting the excitation and emission wavelengths at 675 nm and 720 nm, respectively. Without application of a MF, only a slight fluorescence signal was observed in cancer (Figure 3A). In contrast, a strong D-SMNC-EXO fluorescence signal was detected at the cancer site with application of a MF (1 T) for 24 h, which indicates that D-SMNC-EXOs had a remarkable magnetic-targeting ability. After in vivo imaging, the mice were euthanized, and organs and tumor tissues were harvested for ex vivo imaging. Consistent with the in vivo imaging results, the fluorescence signal in tumors to which the MF had been applied was significantly higher than the control tumor-bearing mice that did not receive MF application (Figure 3B). A quantitative region-of-interest analysis revealed that after MF application, the fluorescence intensity of the cancer increased approximately 2.12-fold (Figure 3C). In general, it was confirmed that D-SMNC-EXOs showed a high accumulation at the tumor site via a combination of passive and active magnetic targeting. Beyond that, the distribution of DOX in major organs and cancer was tested (Figure 3D). D-SMNC-EXOs without external MF changed the biodistribution of DOX and enhanced the targeting ability of DOX through an EPR effect. D-SMNC-EXOs with external MF provided a higher level of targeting delivery efficiency. D-SMNC-EXOs increased the DOX concentration ~1.7-fold in the tumor via a combination of passive and active magnetic targeting. FITC labeled D-SMNC-EXOs were used to analyze the distribution of SMNC-EXOs and DOX in the tumor. The fluorescence signal from the D-SMNC-EXOs in the tumor section with application of external MF (denoted as D-SMNC-EXOs (+MF)) was much stronger than that


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from the D-SMNC-EXOs without application of an external MF (denoted as D-SMNC-EXOs (MF)) (Figure 3E). The fluorescence signals of SMNC-EXOs (green) and DOX (red) did not match completely because part of the DOX was released from D-SMNC-EXOs. The fluorescence level of the SMNC-EXOs does not correlate with that of DOX. These results indicate that D-SMNC-EXOs could accumulate passively at the cancer site and release DOX, and a MF can enhance the active accumulation of D-SMNC-EXOs.

Figure 3. A) Noninvasive NIRF imaging of Cy5.5-labeled D-SMNC-EXOs in Kunming mice after 24 h intravenous injection with/without external MF, B) representative ex vivo NIRF optical images of tumor and major organs, C) radiant efficiency of Cy5.5-labeled D-SMNC-EXOs in


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tumors and major organs, D) levels of DOX in cancers and major organs, E) accumulation of FITC-labeled D-SMNC-EXOs in tumor section was evaluated using fluorescence microscope, green represents SMNC-EXOs and red represents DOX. Significance levels are shown as **p < 0.01 and ***p < 0.001. Tumor suppression by D-SMNC-EXOs. The capacity of D-SMNC-EXOs to suppress cancer in Kunming mice bearing an H22 subcutaneous cancer was evaluated. Mice received intravenous injections on days 10, 13, 16, and 19 with (I) PBS, (II) SMNC-EXOs, (III) free DOX, (IV) D-SMNC-EXOs (-MF), and (V) D-SMNC-EXOs (+MF). In treatment groups, every mouse received ~100 µg DOX (5 mg DOX/kg body weight) or 1 mg D-SMNC-EXOs (with equivalent DOX) per injection. To examine the kinetics of cancer growth, tumor volume was monitored using a caliper before each injection and was calculated as [(length × width2)/2].34 The tumor volumes of mice treated with PBS or blank SMNC-EXOs increased rapidly within three days after injection (Figure 4A). Tumors that received DOX administration showed a slight growth inhibition because of low bioavailability. In contrast, D-SMNC-EXOs (-MF) showed a stronger ability to inhibit tumor growth, which indicates the enhanced bioavailability of DOX as a result of the EPR effect. Tumor growth was suppressed after administration of D-SMNC-EXOs (+MF), which substantiates that the application of an external MF results in enhanced DOX accumulation at the cancer site compared with D-SMNC-EXOs (-MF). The tumor growth kinetics of mice treated with D-SMNC-EXOs (+MF) were 2.9 ± 0.3-fold and 2 ± 0.2-fold those treated with DOX and D-SMNC-EXOs (-MF), respectively. The average sizes of tumors harvested from the mice at day 22 were 4.55, 4.53, 4.15, 2.97, and 1.44 cm3 in mice treated with PBS, SMNC-EXOs, DOX, D-SMNC-EXOs (-MF), and D-SMNC-EXOs (+MF), respectively (Figure 4B). Tumors from


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these mice were ~2.97 g, 2.89 g, 2.53 g, 1.78 g, and 0.76 g, respectively (Figure 4C). This indicates that D-SMNC-EXOs (+MF) produced the most significant anti-tumor effects, which validates the in vivo anti-cancer applicability of D-SMNC-EXOs (+MF).

Figure 4. A) Growth evaluation of H22 subcutaneous cancer in Kunming mice after sample administration, tumor volume was examined every three days for 12 consecutive days, B) H22 tumor tissues obtained from euthanized mice 12 days after sample administration, C) average mass of collected cancer tissues, D) Western blot analysis of apoptosis-related protein expression in tumor tissues 12 days after sample administration, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, significance levels are shown as **p < 0.01 and ***p < 0.001.


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Using Western blot analysis, we examined the expression levels of Caspase-3 and Bcl-2 in cancer tissues harvested from these mice (Figure 4D). D-SMNC-EXOs (+MF) and D-SMNCEXOs (-MF) could inhibit tumor growth and up-regulate apoptosis in H22 cells by decreasing Bcl-2 expression and increasing Caspase-3 expression. The Western blot bands and normalized intensity demonstrate that D-SMNC-EXOs (+MF) administration produced the lowest levels of Bcl-2 expression and highest levels of Caspase-3 expression, respectively (Figure S23), which corresponds to our in vivo findings regarding tumor growth inhibition. Finally, in consideration of the relatively high accumulation of D-SMNC-EXOs in the liver, tests of the serum level of total protein, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase showed that no severe hepatotoxicity resulted (Table 1). Table 1. Effect of SMNC-EXOs and D-SMNC-EXOs on serum levels of total protein, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase

Index

TP (g/L)

ALT (U/L)

AST (U/L)

ALP (U/L)

Control

77.52 ± 8.01

49.16 ± 4.64

144.31 ± 5.72

85.43 ± 4.48

SMNC-EXOs

76.93 ± 5.22

44.64 ± 4.79

140.26 ± 8.72

95.56 ± 5.63

DOX

66.62 ± 3.3

65.45 ± 10.89

160.18 ± 4.08

99.62 ± 20.08

D-SMNC-EXOs (-MF)

72.5 ± 4.29

47.15 ± 4.72

143.13 ± 5.37

85.4 ± 7.42

D-SMNC-EXOs (+MF)

76.97 ± 4.38

44.32 ± 4.11

140.5 ± 20.06

84.59 ± 11.5

Group

TP, total protein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; and ALP, alkaline phosphatase.


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CONCLUSION We have synthesized a dual-functional blood exosome-based superparamagnetic nanoparticle clusters for cancer therapy. Anchoring multiple superparamagnetic nanoparticles onto each RTC-derived exosome through Tf-Tf receptor interaction yields vehicles with superparamagnetic behavior at room temperature, and their response to an external MF is enhanced significantly compared with individual superparamagnetic nanoparticles. By taking advantage of these properties, we have separated exosomes from blood and provided them with a tumor-targeting ability. The drug-loaded exosome-based vehicles show excellent in vivotargeting ability and cancer inhibition effect. However, since Tfs exist in serum, the separate efficiency of RTC-exosomes directly from serum is modest. Replacing Tfs with a unique ligand may enhance the separation efficiency of the RTC-exosomes significantly. The most promising aspect of this study is the strategy of exosome-based SPMN clusters, which can endow the delivery vehicles with superparamagnetic and ferromagnetic characteristics concurrently. This technology can separate and purify exosomes in blood, which can potentially be useful for diagnosing diseases such as cancer. The incorporation of magnetism with exosomes can expand the biomedical applications.

MATERIALS AND METHODS Materials. Carboxyl-group functionalized superparamagnetic Fe3O4 nanoparticles were purchased from Nanjing Nanoeast Biotech Co., Ltd. Nile Red, Doxorubicin (DOX), Fluorescein isothiocyanate (FITC), Cy5.5 fluorescence dye and transferrin (Tf) were purchased from Sigma-Aldrich. BCA protein assay kit was purchased from Thermo Scientific. Hepatoma


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22 cells and Michigan Cancer Foundation-7 were obtained from the China Academia Sinica cell repository (Shanghai, China). Synthesis of M-Tfs. Carboxyl-group functionalized superparamagnetic Fe3O4 nanoparticles solution (40 µL, 2.5 mg/mL) was mixed with EDC and sulfo-NHS at a molar ratio of 1:2:3 (pH 5.5). This reaction mixture was incubated at room temperature for 1 h. Then, 1 µL 2mercaptoethanol was added to terminate the reaction. The activated superparamagnetic Fe3O4 nanoparticles were purified via magnetic separation and were re-suspended in 200 µL Borate buffer (20 mM, pH 8.5). Then, 10 µg holo-transferrin was added, and the mixture was incubated for 12 h at 4°C under nitrogen. Finally, the M-Tfs were purified by magnetic separation and washed three times with PBS. The resulting solution (200 µL) was stored at 4°C until it was used for magnetic separation. Magnetic separation and re-dispersion of SMNC-EXOs. First, 1 mL serum from Kunming mice was dialyzed against PBS for 24 h. Then, the serum solution was mixed with 200 µL M-Tfs solution, and was blended homogeneously using a vortex shaker. This mixture was incubated for 4 h at 4°C. The SMNC-EXOs were obtained after magnetic separation and were washed three times with PBS. To assess the capacity of SMNC-EXOs for re-dispersion, SMNC-EXOs were re-dispersed in 200 µL PBS, and the solution was shaken gently for 10 min. Size and size distribution were measured by dynamic light scattering (BI-90Plus, Brookhaven Instruments Ltd., USA), and the morphology and composition of SMNC-EXOs were visualized using a high-resolution transmission electron microscope (TEM, JEM-2100F, JEOL Ltd., Japan) and subjected to TEM-


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EDS for high-speed elemental analysis. Electron diffraction patterns of samples were also obtained using the TEM. Comparison of magnetic separation efficiency. M-Tfs (200 µL) and SMNC-EXOs (200 µL) were each diluted with PBS to 1 mL, and then added to vials. The vials were placed next to a magnet and the amount of separated magnetic nanoparticles was recorded at selected time intervals. Verification of RTC-derived exosomes. The magnetic separation of RTC-derived exosomes was confirmed by Western blot analysis as described previously with slight modifications.35 Briefly, SMNC-EXOs for Western blots were prepared by lysing exosomes in a radioimmunoprecipitation assay (RIPA) buffer containing 1 mM EDTA. Lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were rinsed with PBS for several minutes and blocked with Odyssey blocking buffer for 1 h at 22°C. The membranes were incubated with primary antibodies against CD9, CD63 (1:1000 dilution; Zhongshan Bio Corp, Beijing, China) and Tf receptor (1:100 dilution; Abcam, Shanghai, China), followed by incubation with fluorescent secondary antibodies (1:1000 dilution; Zhongshan Bio Corp, Beijing, China). Images were acquired with an Odyssey infrared imaging system and analyzed using software specified by the Odyssey systems. As a control group, exosomes, isolated using precipitant-ExoQuick, were manipulated according to the abovementioned method. Demonstration of formation mechanism of SMNC-EXOs. SPMNs (40 µL, 2.5 mg/mL) were incubated with 1 mL pre-dialyzed blood serum for 4 h at 4°C and then the mixture was subjected to magnetic separation. Then size and size distribution of the obtained particles were


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measured by dynamic light scattering, and the morphology was visualized using a highresolution transmission electron microscope. To test the protein concentration of SMNC-EXOs at different pH, we firstly obtained fresh SMNC-EXOs. Then SMNC-EXOs were averaged into three groups. The first and second groups were placed in pH 7.4 and pH 5.0 buffer solutions, respectively. Both groups were cultivated for 24 h. The third group was placed in buffer solution (pH 5.0) for 12 h and then the buffer solution pH was adjusted to 7.4. SMNC-EXOs were incubated in pH 7.4 buffer for a further 12 h. After incubation, the SMNC-EXOs were separated magnetically. Protein concentrations of the three groups were measured using a BCA Protein Assay according to the manufacturer's instructions. Samples were reacted with working reagent at 37°C for 30 min, and the absorbance was then measured at 562 nm. Values were compared with a bovine serum albumin standard curve. Stability of SMNC-EXOs. SMNC-EXOs were transferred into glass vials to incubate at 4°C in PBS buffer and 37°C in serum. At selected time intervals, SMNC-EXOs in serum were separated magnetically and were re-dispersed in PBS buffer. The particle size of two samples was evaluated using dynamic light scattering. Measurements of the two groups were taken in sextuplicate, and results were averaged. To study the influence of serum Tfs on the stability of SMNC-EXOs, they were incubated with fresh serum. After incubation at 37°C for 24 h, SMNCEXOs were separated magnetically and were re-dispersed in PBS buffer. The concentration of SMNC-EXOs was measured using BCA protein assay. To compare SMNC-EXOs with MB-EXOs, 1 mL Exosome-Human CD63 Isolation/Detection Reagent (Life Technologies, USA) and 200 µL M-Tfs was mixed with 1 mL pre-dialyzed serum. After incubation for 24 h, MB-EXOs and SMNC-EXOs were separated


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magnetically and were re-dispersed. The size and size distribution were measured by dynamic light scattering, and the stability was observed and recorded. In vitro evaluation of magnetic targeting ability of SMNC-EXOs. A blood circulation system was simulated using a microfluidic system as designed in Figure S12. M-Tfs and SMNCEXOs (0.1 mg/mL) were added into the circular flow, and the liquid flow rate was controlled by gas pressure and held at 32.85 cm/s (artery), 14.60 cm/s (vein), and 0.05 cm/s (capillary). In addition, a magnet was placed under the pipe. After a few minutes, magnetic retentions of M-Tfs and SMNC-EXOs were observed and recorded. Detection of hydrophobic domain of SMNC-EXOs. Nile Red solution (2 µL, 2 mg/mL) was added to each vial containing SMNC-EXOs solutions of different concentrations (0, 56.5, 113, 226 and 452 µg/mL). The same concentrations of BSA solution were regarded as the control, and 2 µL Nile Red was added to each BSA solution. After samples had been incubated for 4 h at 4°C, the fluorescence intensity was measured on a spectrophotometer. According to the pre-scan of excitation and emission characteristics of lipid standards, excitation and emission wavelengths of 530 nm and 575 nm were selected. DOX loading. For DOX loading, 20 µL DOX hydrochloride solution (2 mg/mL) was added to the SMNC-EXOs solution (200 µL, 1 mg/mL) with moderate stirring. After 30 min, 5 µL triethylamine was added, and the solution was stirred a further 1 h. D-SMNC-EXOs were obtained via magnetic separation at 4°C. The amount of DOX loaded into SMNC-EXOs was calculated from a calibration curve acquired from UV-vis spectrophotometer measurements based on the absorbance intensity at 485 nm. The size and size distribution of D-SMNC-EXOs were measured by dynamic light scattering.


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In vitro drug release. The release of DOX was performed under two conditions, namely, with PBS (pH 7.4) and acetate buffer (pH 5.0) as described previously with slight modifications.36 In short, 4 mL D-SMNC-EXOs solution was transferred into a dialysis tube (molecular weight cut-off: 14 kDa). The tube was placed into a 10 mL buffer. The release of DOX was performed at 37°C in a water box at a constant temperature. At selected time intervals, the dialyzate was removed for UV-vis spectrophotometer analysis and replaced with a fresh buffer solution. DOX concentrations were determined according to standard curves at the corresponding buffer solutions. Cell viability assay. The cytotoxicity of SMNC-EXOs in H22 cells was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Firstly, 4000 cells were seeded into 96-well plates and grown in complete medium containing L-DMEM medium,10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine at 37°C for 24 h. Subsequently, the culture medium in each well was replaced with fresh medium that contained SMNC-EXOs in a series of concentrations. Cells in wells without the addition of SMNC-EXOs were used as a control group. Each group included six replicates. After culturing for a further 48 h, CCK solution was added and cell viability was calculated as the ratio of the absorbance of test and control wells. The absorbance was measured at 450 nm with a reference at 650 nm using a microplate reader (Synergy2, Bio-Tek, USA). Cellular uptake. D-SMNC-EXOs were labeled with FITC and different concentrations of the fluorescent labeled sample were added to H22 cells and incubated for 4 h at 37°C. The samples were washed three times with PBS at the corresponding temperature, and FITC signal uptake rates were detected using flow cytometry (Becton, Dickinson and Company, USA).


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According to the above method, FITC signals of D-SMNC-EXOs (-MF) and D-SMNC-EXOs (+MF) uptake rates and DOX signals of DOX and D-SMNC-EXOs uptake rates were detected. Confocal fluorescence microscopy was used to assess intracellular trafficking of SMNCEXOs and DOX. Cells that had grown on the glass coverslips (pretreated with polylysine) of a six-well plate were incubated with DOX, FITC-labeled SMNC-EXOs and FITC-labeled DSMNC-EXOs for 24 h, respectively. Following incubation, the cells were washed three times with PBS and were fixed in paraformaldehyde for 15 min. Localization of FITC-SMNC-EXOS and DOX in cells was visualized using a confocal microscope (Carl Zeiss Microscope Systems, Jena, Germany) with identical settings for each confocal study. Inhibition of cancer cells by D-SMNC-EXOs. Cancer suppression effects of DOX and DSMNC-EXOs were evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Firstly, 4000 H22 cells were seeded into 96-well plates and grown in complete medium containing LDMEM medium,10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine at 37°C for 24 h. Subsequently, the culture medium was replaced with complete medium containing 0, 0.25, 0.5, 1, 2, 4, 8, 10 and 12 µg/mL DOX. The same procedure was carried out to study how D-SMNC-EXOs influenced cell viability, with 0, 0.25, 0.5, 1, 2, 4, 8, 10 and 12 µg/mL DOX equivalent concentrations. At 48 h, CCK solution was added and cell viability was assessed. Unexposed wells were regarded as the control, and cell viability was calculated as the ratio of the absorbance of the test and control wells. The absorbance was measured at 450 nm with a reference at 650 nm using a microplate reader (Synergy2, Bio-Tek, USA).


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Blood compatibility assay. Fresh heparin-stabilized mice blood was collected. A 4 mL sample of whole blood was added to 8 mL of saline, and red blood cells were isolated by centrifugation at 1,000 g for 15 min. RBCs were washed five times with sterile saline solution. Following the final wash, the RBCs were diluted with 40 mL of saline. Then, 0.2 mL of the diluted RBC suspension was added to 0.8 mL of SMNC-EXOs to achieve final SMNC-EXOs concentrations of 10, 100, and 1,000 µg/mL. The suspension was vortexed briefly before leaving it under static conditions at room temperature for 4 h. Thereafter, the mixture was vortexed briefly again and centrifuged at 1,000 g for 10 min. Next, 400 µL supernatant was measured using UV-vis absorbance spectrum scanning. After that, 0.2 mL of diluted RBC suspension, which was incubated with 0.8 mL of saline and 0.8 mL of distilled water, was used as the negative or positive control. Histopathological evaluation of major organs. Kunming mice, four to six weeks old, were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. The mice were divided randomly into two groups: one group was injected intravenously with a solution of SMNC-EXOs (5 mg/mL, 200 µL per mice), whereas the control group was injected with PBS. After 48 h, the mice were euthanized, and major organs were harvested. All experimental protocols were conducted within Tianjin Medical University’s guidelines for animal research and were approved by the Institutional Animal Care and Use Committee. The organs were stored overnight in 2.0% (V/V) formaldehyde solution in PBS and were then washed twice with PBS to remove excess formaldehyde. Paraffin-embedded tissue sections were stained with hematoxylin andeosin (H&E) and observed through a microscope. Biodistribution and magnetic targeting for cancer in vivo. D-SMNC-EXOs were labeled by NHS-CY5.5 (mass ratio of 100:1) in pH 8.5 buffer solution. Following incubation for 4 h,


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CY5.5-labeled D-SMNC-EXOs were separated magnetically and the supernatant was removed. The separated samples were washed three times with PBS and were re-dispersed into PBS. Kunming mice, four to six weeks old, were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. H22 cells were suspended in serumfree DMEM medium and inoculated subcutaneously to the flanks of mice (2 × 106 cells per mice). After cancers had grown to ~100 mm3, the mice were divided randomly into three groups. One group was injected intravenously with Cy5.5-labeled D-SMNC-EXOs solution (5 mg/mL, 200 µL per mice). Then, a magnet (MF density: 1 T) was placed over the surface of the cancer using Steri-Strip tape. One group was injected intravenously with Cy5.5-labeled D-SMNC-EXOs solution without magnet. The control group was injected with PBS. After 24 h, the magnet was removed from the mice. Whole-animal imaging was recorded using an IVIS Spectrum imaging system (IVIS 100, USA). After that, the mice were euthanized, and the cancers and major organs were harvested, washed with PBS, and placed in a dish. Next, fluorescence imaging results and average radio intensities were recorded using an IVIS Spectrum imaging system. To investigate the change in biodistribution of DOX by D-SMNC-EXOs, free DOX and DSMNC-EXOs without and with external MF (5 mg of DOX-equiv. per kg of body weight) were injected intravenously into mice with cancer. After 4 h, the mice were euthanized, and the cancers and major organs were harvested, washed with PBS, stored in liquid nitrogen and triturated in mortar. The powder was then dissolved in 1 mL borate buffer solution and ultrasound lysed. After 30 min, 1 mL chloroform was added to the solution, and the mixed solution was shaken for 30 minutes. Finally, the solution was allowed to remain stationary and the lower solution was absorbed. According to the absorption standard curve of DOX, the absorbance was measured at 480 nm to determine the DOX content.


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To observe the distribution of SMNC-EXOs and DOX in cancer tissue, FITC-labeled DSMNC-EXOs solution (5 mg/mL, 200 µL per mice) was injected intravenously into mice with cancer. There were three groups: a control group, FITC-labeled D-SMNC-EXOs without magnet and FITC-labeled D-SMNC-EXOs with magnet. After 24 h, the magnet was removed from the mice. Isolated cancer tissues were embedded in optimal cutting temperature (OCT) compound and were frozen rapidly to −20°C for 24 h. Tumor tissues were cut into 8 mm histology slices using a cryostat. Each section was dyed with 4',6-diamidino-2-phenylindole (DAPI) and covered with a coverslip. The frozen sections were observed using an IX51 fluorescence microscope from Olympus Corpoartion (Tokyo, Japan). In vivo anticancer efficacy study. Kunming mice, four to six weeks old, were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. H22 cells were suspended in serum-free DMEM medium and were inoculated subcutaneously to the flanks of mice (2 × 106 cells per mice). After cancers had grown to ~100 mm3, the mice were divided randomly into five groups: PBS, DOX, SMNC-EXOs, D-SMNC-EXOs without application of a magnetic field, and D-SMNC-EXOs with application of a magnetic field. Solutions were administered by intravenous injections every three days (5 mg of DOX-equiv. per kg of body weight per dose) for two weeks. Mouse weights and cancer mass were measured. The cancer volume was measured from: volume = length × width2/2. The mice were euthanized, and the cancers were harvested. We photographed the cancers and measured their average masses. The cancers were frozen in liquid nitrogen. Protein extracts were made by grinding cancers in 100 ml of grinding buffer. Total protein was quantified using a NanoDrop 2000 (Thermo Scientific). Protein lysates were separated using SDS-PAGE gel and were transferred onto PVDF membranes, then incubated with primary antibodies that could detect Caspase-3 and Bcl-2


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(1:1000 dilution, Zhongshan Bio Corp, China), followed by incubation with a secondary antibody (1:1000 dilution, Zhongshan Bio Corp, China). GAPDH was selected as a control. Protein expression levels were quantified by normalized gray values using ImageJ software. Serum cytokine measurement. The levels of total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in serum were determined using an automatic serum chemical analyzer. The concentrations of each cytokine in the serum were determined using commercially available ELISA kits (eBioscience, USA). Briefly, each microplate well was coated with 100 µl of capture antibody, and incubated overnight at 4°C. After washing and blocking with assay diluent, serum or standard cytokines were added to individual wells and the plates were maintained for 2 h at room temperature. The plates were washed, and biotin-conjugated detecting mouse antibody was added to each well and incubated at room temperature for 1 h. The plates were washed again and were incubated further with avidin–HRP for 30 min before detection with TMB solution. Finally, reactions were stopped by adding 1 M H3PO4, and the absorbance at 450 nm was measured with an ELISA reader (Molecular Devices, USA). The amount of cytokine was calculated from the linear portion of the generated standard curve. Statistical analysis. Results are presented as mean ± SEM. Statistical comparisons were performed by paired t tests with a two-tailed p value to compare selected data pairs when only two groups were compared, and if more than two groups were compared, an evaluation of significance was performed using a one-way analysis of variance (ANOVA) followed by a Dunnett post-test using GraphPad Prism 5.0 software.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Acknowledgements. This work was supported by grants from the National Nature Science Foundation of China (Grant Nos. 51303125 and 51473119) and the National High Technology Research and Development Program of China 863 (Grant Nos. 2014AA021102 and 2012AA02A508).

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Blood Exosomes Endowed with Magnetic and ... - ACS Publications

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