In situ single step detection of exosome microRNA using molecular beacon

Biomaterials 54 (2015) 116e125

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In situ single step detection of exosome microRNA using molecular beacon Ji Hye Lee a, 1, Jeong Ah Kim b, 1, Min Hee Kwon a, Ji Yoon Kang c, Won Jong Rhee a, * a

Division of Bioengineering, Incheon National University, Incheon 406-772, Republic of Korea Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA c Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2014 Received in revised form 6 March 2015 Accepted 9 March 2015 Available online 31 March 2015

In situ single step detection of microRNAs (miRNA) in a whole exosome has been developed as a novel diagnosis method that can be utilized for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs produced from their originating cells and are known to travel through the circulatory system. This makes exosomal miRNAs from the body fluids an attractive biomarker that can lead to a paradigm shift in the diagnosis of disease. However, current techniques, including realtime PCR analysis, are time-consuming and laborious, making them unsuitable for exosomal miRNA detection for diagnosis. Thus, the development of alternative methods is necessary. Herein, we have demonstrated that exosomal miRNAs can be detected directly using a nano-sized fluorescent oligonucleotide probe, molecular beacon. MiRNA-21 in exosomes from breast cancer cells were detected successfully by molecular beacons in a quantitative manner. Permeabilization by streptolysin O treatment further enhanced the delivery of molecular beacons into exosomes, giving significantly increased signals from target miRNAs. In addition, we selectively detected cancer cell-derived exosomal miRNA-21 among heterogeneous exosome mixtures and in human serum. The method developed in the article is simple, fast, and sensitive, so it will offer great opportunities for the high-throughput diagnosis and prognosis of diseases. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Exosome Molecular beacon microRNA Cancer Diagnosis

1. Introduction Exosomes are small extracellular vesicles with sizes of 30e100 nm that are produced and secreted by a wide variety of different cells in the body [1e4]. They are known to enter the circulatory system, and, therefore, they are found in most of body fluids, including blood, urine, semen, saliva, bile, and breast milk [5e7]. In the past, it has been recognized that exosomes have a key role in the elimination of unnecessary biomolecules, such as proteins, from cells. However, current views on exosomes are drastically different, and their physiological and pathological significance has been increasingly highlighted. They are now thought of as extracellular organelles that contain important cellular biomolecules and that mediate intercellular communications among cells and tissues in the human body by conveying information to

* Corresponding author. Tel.: þ82 32 835 8299; fax: þ82 32 835 0763. E-mail address: [email protected] (W.J. Rhee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.03.014 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

distant tissues, thereby regulating, for example, the proliferation, differentiation, death, and immune response of cells [5,6,8e10]. Similar to cells, exosomes are encompassed with lipid bilayer and composed of proteins, peptides, lipids, carbohydrates, and nucleic acids derived from their originating cells [11]. Thus, detection of exosomal biomolecules from the body fluids offers great opportunities for the diagnosis of diseases. MicroRNAs (miRNAs) are small non-coding RNAs of 19e25 nucleotides in length, and they have key roles in regulating cellular processes, such as development, differentiation, proliferation, and apoptosis in the human body [12]. There has been extensive research on utilizing miRNAs as biomarkers for the identification of diseases, because miRNAs reflect an important pathogenic process [13e21]. For example, the high expression level of miRNA-21 (miR21) is known to be associated with low survival rate and poor therapeutic outcome of colon adenocarcinoma [22]. Recent studies have shown that miRNAs also are present in circulating exosomes and transferred to other cells, altering the function of those target cells [1,23]. These findings provide evidence that exosomal miRNAs can be used for the early prediction of disease signature in the body

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and, in this context, specific exosomal miRNAs have proven to be important biomarkers of diseases [24]. Different miRNAs are involved in and can be used as diagnostic biomarkers of various cancers, i.e., lung, breast, pancreatic, colon, metastatic gastric, prostate, ovarian, and esophageal cancers [23,25e31]. In most cases, the current methods for the detection of miRNA from the exosomes require a series of procedures, even after isolation of the exosomes, ranging from sample treatment to detection, such as exosome lysis, miRNA isolation, cDNA synthesis, and real-time PCR analysis. In addition, due to its small size, mature miRNA must be elongated with a specific oligonucleotide, such as a poly (A) tail, before the cDNA synthesis step to ensure that the size is large enough to be detected in the PCR product. Thus, it is essential to have an easy, simple, accurate, fast, and inexpensive technique for the detection of specific miRNAs in exosomes. The molecular beacon (MB), nano-sized oligonucleotide probe is dual-labeled oligonucleotide hairpin probe with a fluorophore and a quencher at each end. This unique structure makes the MB a suitable probe for the imaging of RNAs in living cells, because it has high specificity and low background fluorescence [32e38]. Unbound MBs that are not interacting with the target RNAs in the living cells do not have to be removed from the cells due to their self-quenching ability [39]. Recently, miRNAs in solution were successfully quantified by using MBs, and MBs that targeted mature or premature miR-21 had high specificity and also distinguished the small fold changes of miRNA expression similar to real-time PCR analysis [40]. Herein, we used MBs for the in situ detection of specific miRNAs from exosomes. In this study, we isolated exosomes from MCF-7, i.e., breast cancer cells, and we detected exosomal miR-21 as a target miRNA, because it is known to be a key player in many diseases, including cancer of the lung, breast, and colon (Fig. 1). We tested exosomes that originated from different types of cells to determine whether MBs bind to miR-21 with high specificity. We also investigated whether MB is delivered into exosomes by going through the exosomal membrane and discussed whether permeabilization treatment can be used to improve the delivery of MBs inside the exosome, giving a high level of hybridization.

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2. Materials and methods 2.1. Detection of miR-21 using MB in solution The design of MB used in this study for targeting mature miR-21 was 50 -Cy3GCGCGTCAACATCAGTCTGATAAGCTACGCGC-BHQ2-30 [40]. The random MB (R-MB) sequence, i.e., 50 -Cy3-GCGCGTCTCTGCCTAAGTGATGTCACGCGC-BHQ2-30 , was used as a negative control. The underlined bases indicate the stem sequence of MB. MiR21 MB and R-MB were synthesized by Integrated DNA Technologies and Cosmogenetech, Inc. respectively. Sequences of synthetic target miR-21 DNA, random target DNA for solution assay, and blocking DNA were 50 -TAGCTTATCAGACTGATGTTGA-30 , 50 -TCAACATCAGTCTGATAAGCTA-30 , and 50 -CAACATCAGTCTGATAAGCT-30 , respectively (Cosmogenetech, Inc.). Hybridization of MB with target miRNA in PBS solution was assessed using a Varioskan™ Flash Multimode Reader (Thermo Scientific, USA) by measuring the fluorescence signals with an excitation wavelength of 545 nm and an emission wavelength of 570 nm. For the reaction, the samples were incubated at 37  C for up to 2 h in a black 384-well microplate until they were to be analyzed. 2.2. Cell culture and exosome-free FBS preparation MCF-7 cells and NIH/3T3 cells were cultured in DMEM (Biowest, France), and CHO-K1 cells were cultured in IMDM (Life Technologies). All cell lines were maintained in a humidified atmosphere of 5% CO2 at 37  C, and all media were supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest) and 1% (v/v) penicillin and streptomycin (Life Technologies). For the production of exosomes from the cells, initially, the cells were grown in media that contained 10% FBS to 70% confluence, washed twice with PBS, and then maintained for an additional two days in media that contained 10% exosome-free FBS. For the preparation of exosome-free FBS, the FBS was loaded into polycarbonate tubes and centrifuged at 4  C at 120,000 g for 10 h using a TLA-100.3 fixed angle rotor in an ultracentrifuge (Optima TL-100, Beckman Coulter, U.S.). The supernatant was filtered using a 0.22-mm syringe-filter and stored at 4  C. 2.3. Exosome isolation by Total Exosome Isolation™, ExoQuick-TC™, and ultracentrifugation In our research, three different methods were used for exosome isolation, two of which were commercially available reagents from different companies. The exosomes were isolated from culture media after cell culture according to the manufacturer's instruction. Briefly, the conditioned media were centrifuged at 2000 g for 30 min to remove cell debris. Next, a volume of the supernatant was mixed with an equal volume of Total Exosome Isolation™ solution (Invitrogen) and mixed well. Then, the mixture was incubated at 4  C overnight and then centrifuged at 10,000 g for 1 h. The supernatant was discarded, and the pellets that contained the exosomes were resuspended in PBS. The exosomes also were isolated by a precipitation method using ExoQuick-TC™ (System Biosciences) according to the vendor's instructions although we did not show the results using exosomes isolated using ExoQuick-TC™ as they are similar to

Fig. 1. Illustration for the in situ detection of exosomal miR-21 using MB for the diagnosis of diseases such as cancer.

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those from Total Exosome Isolation™. The conditioned media were centrifuged at 3000 rpm for 15 min, and the supernatant was incubated overnight with ExoQuickTC™ solution. The next day, the mixture was centrifuged at 3000 rpm for 30 min, and the pellets were resuspended in PBS. In addition to the commercially available reagents listed above, exosomes were isolated by differential ultracentrifugation (Optima TL-100, Beckman Coulter, U.S.). Briefly, conditioned media were centrifuged at 4  C at 300 g for 10 min and then at 2000 g for 10 min to remove cell debris. Then, the media were centrifuged at 4  C at 10,000 g for 30 min to produce cell-free conditioned media. The conditioned media were divided equally into six polycarbonate tubes and centrifuged at 4  C at 100,000 g for 2 h using a TLA-100.3 fixed angle rotor (Beckman Coulter). The subsequent pellets were resuspended and washed with PBS, followed by a second 100,000 g centrifugation. The final pellets, which consisted of purified exosomes, were resuspended in PBS for later use. 2.4. Exosome quantification by ELISA and BCA assays The exosomes that were isolated were analyzed to quantify their protein contents using BCA assay (Thermo Scientific). In addition, to quantify the amount of exosome particles released, we conducted ELISA using an Exosome ELISA kit (System Biosciences). Briefly, the isolated exosomes were added and incubated overnight in the micro-titer plate provided in the kit. Standard exosomes with known concentration supplied in the kit also were run on ELISA. After three washes with washing buffer, exosome-specific anti-CD63 (System Biosciences) was added and incubated for 1 h. After washing, the plate was incubated with HRP-conjugated anti-rabbit antibody (System Biosciences) for 1 h. After the final washing, the reaction was developed with super-sensitive TMB substrate followed by blocking with a stop buffer, and optical densities were recorded at 450 nm using a Varioskan™ Flash Multimode Reader. 2.5. Western blot analysis of exosomal protein Cells and exosomes were lysed in RIPA buffer (Rockland), and the protein concentrations were determined using a BCA protein assay kit. All SDS-PAGE were conducted using 12% polyacrylamide separating gels. The gels were transferred to a PVDF membrane in Towbin transfer buffer (197 mM glycine, 25 mM Tris (pH 8.3), 0.037% SDS, 20% methanol), at 100 V for 1 h. After blocking with 5% skim milk in TBS (pH 7.4) that contained 0.1% Tween 20, the membrane was incubated with the rabbit anti-human CD63 antibody (System Biosciences), and this was followed by incubation with an HRP-conjugated secondary antibody. An ECL detecting system (BioRad, U.S.) was used to visualize the immunoreactive bands, and the images were obtained using a KODAK Image Station 4000R (Carestream Health, Inc., Rochester, NY, U.S.). 2.6. Transmission electron microscopy (TEM) The exosomes isolated by ultracentrifugation were resuspended in PBS and then absorbed onto a formvar carbon-coated grid for 10 min. After washing the grid with distilled water, exosomes were fixed in 2% paraformaldehyde and washed twice with PBS. Then, the grids were negatively stained with 2% uranyl acetate for 10 min. Samples were dried for 15 min and visualized using a JEM-1010 electron microscope (JEOL, Japan) operated at 60 kV. 2.7. RNA isolation, cDNA synthesis, and real-time PCR analysis A conventional assay of miRNA was performed using real-time PCR. For the preparation of samples, total RNA from exosomes and cultured cells were isolated using the ISOL reagent (5PRIME) according to the manufacturer's protocol. The concentrations of all RNA samples were quantified using the NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, U.S.). Then, total RNA was reverse-transcribed to cDNA using a miScript RT II kit (Qiagen, Hilden, Germany) that contained 4 mL of 5 miScript RT buffer and 2 mL of miScript Reverse Transcriptase Mix. The reactions were incubated at 37  C for 60 min, at 95  C for 5 min, and then held at 4  C. The mature miR-21 levels were measured by quantitative realtime PCR analysis using a miScript SYBR green PCR kit (Qiagen, Hilden, Germany). The reactions were performed and analyzed using ABI PRISM 7300 SDS software. The PCR mixture included 10 mL of 2 QuantiTect SYBR Green PCR Master Mix, 2 mL of 10 miScript Universal Primer, 2 mL of 10 miScript Primer Assay (specific for miR-21 and RNU6B, Qiagen, Hilden, Germany), and 4 mL of RNase-free water. The PCR conditions were as follows: 95  C for 15 min, followed by 40 cycles of 94  C for 15 s, 55  C for 30 s, and 70  C for 35 s. 2.8. Detection of exosomal miR-21 using MB A certain amount of exosomes was incubated with MBs for the detection of exosomal miR-21. The concentration of exosomes was determined by an exosome ELISA kit (System Biosciences). The exosome and MB mixture into PBS were incubated for 1e2 h at 37  C while being protected from light. The concentrations of exosome and MB used are described in the results section for each experiment. Measurement of the signal-to-noise ratio of the molecular beacons was conducted every hour using a Varioskan™ Flash Multimode Reader.

2.9. MB delivery into exosomes using streptolysin O (SLO) The MBs were delivered into exosomes with the reversible permeabilization method using activated streptolysin O (Sigma, U.S.). Specifically, SLO was activated first by adding 5 mM of tris(2-carboxyethyl)phosphine (Thermo, U.S.) to 2 U/mL of SLO for 1 h at 37  C. The exosomes/MB/SLO mixture in PBS was incubated for 1e2 h at 37  C for MB delivery and hybridization. 2.10. Detection of exosomal miR-21 in human serum For the detection of exosomal miR-21 in human serum, certain amounts of exosomes were spiked into human serum (Sigma H4522, U.S.). The concentration of exosomes in the serum was determined by an exosome ELISA kit (System Biosciences). 3  1010 particles/mL of exosomes from MCF-7 cells were added to 35 mL of human serum, followed by the addition of 1 mM of MB. The total hybridization volume was 50 mL. Fluorescence signals were measured 1 h after the reaction began using a Varioskan™ Flash Multimode Reader.

3. Results 3.1. Detection of miR-21 using MB in solution To test the specificity of miR-21 targeting MB, we incubated MB with synthetic target DNA, which has the same deoxynucleotide sequence as mature miR-21 and measured its signal-to-background (S/B) ratio after the hybridization reaction. The background signal indicates the fluorescence intensity from MB without target miRNA. As expected, increased concentrations of target miR-21 had higher S/B ratios, while the highest concentration (100 nM) of random target DNA (RT 100), which does not have a complementary sequence to MB, had a very low S/B ratio (Fig. 2A and B). The S/ B ratio for even the lowest concentrations (between 100 pM and 1 nM) of target miR-21 also increased gradually, showing the high sensitivity of MB. Random MB, which was designed not to bind to miR-21, did not show any increased signals even with high concentrations of miR-21 (Fig. 2C and D). Therefore, it was obvious that miR-21 targeting MB had high specificity and sensitivity. 3.2. Production, isolation, and characterization of exosome from MCF-7 cells The breast cancer cell line, MCF-7, was cultured with exosomefree FBS for two days, and the exosomes that were produced were isolated. Although we used and tested exosomes that were isolated by three independent methods, i.e., ultracentrifugation, Total Exosome Isolation™, and ExoQuick-TC™, the majority of the experiments were conducted with exosomes from Total Exosome Isolation™. To confirm the efficacy of the isolation of the exosomes, we performed the Western blot analysis against CD63, an exosome marker that is localized at the surface of the exosome, and observed the existence of CD63 in a dose-dependent manner (Fig. 3A). Dual band of CD63 from exosomes was shown as reported previously [30]. Electron microscopic analysis of isolated exosomes also showed round structures with sizes varying from 30 to 100 nm, as shown in Fig. 3B, which was consistent with the previously reported characteristics of exosomes [1e4]. 3.3. Real-time PCR analysis of miRNA-21 in cells and exosomes To assess the level of miR-21 in MCF-7 cells and their derivatives, exosomes, we extracted miRNAs from the cells and exosomes and then measured their relative amounts of miR-21 using real-time PCR analysis. MiR-21 is known to be expressed in humans and in other species, such as mice and hamsters. Thus, as a comparison, we also analyzed the miR-21 level of CHO-K1 cells and exosomes. The same amount of RNAs from cells and exosomes were used for real-time PCR. Li et al. have reported that approximately 1e3  1012 exosomes can be isolated from 1 mL of serum [30]. In addition,

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Fig. 2. Detection of miR-21 using MB in solution: (A, B) MB targeting miR-21 was incubated with different concentrations of synthetic miR-21, and 5 nM MB was used for the detection of lower concentrations of miR-21 (A) incubated for 2 h and 10 nM was used for higher concentrations (B) incubated for 30 min. RT 100 indicates 100 nM of random target DNA, which does not have complementary sequence to MB. No signal increase was detected even when the high concentration RT 100 was used. (C, D) Random MB was also used for the hybridization assay with synthetic miR-21, and no signal differences were observed from low to high concentrations of miR-21. All values are mean ± SD (*p < 0.05, **p < 0.01, *** p < 0.001; n ¼ 3e5).

Fig. 3. Characterization of isolated exosomes from MCF-7 breast cancer cells and quantification of miR-21 in MCF-7 breast cancer cells and their exosomes. (A) Exosomes were purified using ultracentrifugation and lysed. Ten and 20 mg of exosomal proteins were used for the detection of exosomal protein marker, CD63, by Western blotting. (B) Exosomes purified were observed for their morphological characterization under transmission electron microscopy (scale bar equals 100 nm). (C, D) Real-time PCR analysis was conducted for cellular (C) and exosomal (D) miR-21, respectively. Exosomes were produced from MCF-7 or CHO-K1 cells and then purified by Total Exosome Isolation™. The same amount of RNAs from each cells and exosomes was used for real-time PCR analysis for comparison. For exosomes, same amount RNAs equals to 4.26  1011 (2.13  1010 particles/mL) and 3.77  1011 (1.89  1010 particles/mL) exosomes for MCF-7 and CHO-K1, respectively. The miRNA levels in MCF-7 cells or their exosomes were normalized by that in CHO-K1 cells or their exosomes, respectively.

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exosomes derived from 4 mL of serum typically yielded 2e10 ng of RNA. Based on this literature, the amount of RNA in one exosome is approximately 1.67  1013e2.50  1012 ng/exosome. From our measurement, total RNA amount in MCF-7 cells was 0.007 ng/cell while that in exosomes from MCF-7 cells was 5.71  1012 ng/ exosome. Thus, real-time PCR analysis using the same amount of RNAs is to compare the relative concentration of miR-21 between two different exosomes or cells but not between exosomes and cells. As shown in Fig. 3C, miR-21 was highly expressed in the MCF-7 cells, which was consistent with previous reports in which miR-21 was deemed to be a potential biomarker for cancer [15,16,22], and the level was 10 times higher than that in the CHO-K1 cells. Then, we measured the exosomal miR-21 level in MCF-7 and CHO-K1 exosomes. The amount of exosomal miR-21 in MCF-7 exosomes was extremely higher (213 times as much) than that in CHO-K1 exosomes (Fig. 3D). Considering the number of exosomes from both cell lines used for the PCR reaction, the difference was about 189 times higher in the MCF-7 exosomes than in the CHO-K1 exosomes. It was interesting to observe that fold difference of miR-21 in the exosomes produced from the two cell lines was much greater than that in the cells, i.e., 213 times and 10 times as much, respectively. This can be interpreted to indicate that exosomal miRNAs are potentially excellent candidates to serve as biomarkers for disease detection since miRNAs from cancer cells are more concentrated in exosomes. Real-time PCR of U6 snRNA (small nuclear RNA) was also carried out, and miR-21 levels in cells (Fig. S1A) and exosomes (Fig. S1B) were normalized by U6 snRNA instead of total RNA, respectively. The amount of exosomal miR-21 in MCF-7 exosomes was still much higher (90 times as much) than that in CHO-K1 exosomes. 3.4. Direct detection of exosomal miR-21 using MB Based on the results provided above, we used MB targeting miR21 for the direct detection of exosomal miRNAs without any additional processes. A hundred nM of miR-21 targeting MB was incubated with different concentrations (0e6  1010 exosomes/mL) of exosomes to determine whether MB can detect the existence of miR-21 inside the exosomes. Interestingly, we observed significantly increased fluorescence intensities within 1 h of the hybridization reaction, and the S/B ratio increased gradually, corresponding to the concentration of exosomes, meaning that the hybridization of MB with exosomal miR-21 was successful (Fig. 4A). Exosomes from CHO-K1 cells also were isolated using the same method for comparison. As shown in Fig. 4B, the S/B ratio from CHO-K1 exosomes increased slightly as the concentration of exosome increased because there were target miR-21s in the exosomes, but their concentration was much lower than that in the MCF-7 exosomes. These results were consistent with the realtime PCR results shown in Fig. 3D, in that the concentration of miR-21s in the MCF-7 exosomes was much higher than that in the CHO-K1 exosomes. We have additionally isolated exosomes from NIH/3T3 mouse fibroblast cells as another negative control and S/B ratio was very low even though high concentrations of exosomes were used for exosomal miR-21 detection (Fig. S2). We can infer from those results shown above that the fluorescence is mainly from the hybridization of MB with exosomal miR-21 but not from non-specific MB and exosome interaction. Therefore, we can conclude that MB can successfully detect specific miRNAs inside the exosomes. It was also suspected that the exosomes were damaged during isolation, thereby releasing intra-exosomal components, including miR-21, into the solution. These released miRNAs in the solution can participate in the hybridization reaction with MB and

Fig. 4. Detection of miR-21 using molecular beacons in whole exosomes. Exosomes were produced from each cell line and purified by Total Exosome Isolation™. The indicated amounts of exosomes were incubated with MBs for 1 h for the detection of exosomal miR-21. (A, B) Exosomes from MCF-7 (A) and CHO-K1 (B) cells were used for the intra-exosomal hybridization reaction of MB and miR-21, respectively. Background signal indicates the fluorescence intensity from MB without exosomes and S/B ratio of each reaction was normalized to control (without exosomes). All values are mean ± SD (*p < 0.05, ***p < 0.001; n ¼ 3).

produce high fluorescence. To exclude this possibility, we synthesized blocking DNA, which has the same sequence with the loop of MB targeting miR-21, but without the stem, fluorescent dye, and quencher. First, we tested the hybridization efficiency of blocking DNA to synthetic miR-21, resulting in the inhibition of MB hybridization in solution before use in our exosome experiments (Fig. 5A). A hundred nM of synthetic miR-21 was incubated with 500 nM of blocking DNA before addition of MB. After 1 h of pre-incubation, 100 nM MB was added to the reactant and incubated for another hour before analysis. Hybridization of MB with miR-21 in solution significantly increased, as was shown in Fig. 5A. And pre-incubation of blocking DNA before adding MB completely eliminated the fluorescence signals, demonstrating that blocking DNA competed with MB and inhibited the hybridization. Based on this, we used this blocking DNA to investigate whether the fluorescence signals shown in Fig. 4A originated from the intraexosomal hybridization of MB with miR-21. If miR-21s were released from damaged exosomes, blocking DNA can bind to them in the solution and compete with MB, resulting in a decrease in fluorescence. Five hundred nM of blocking DNA was preincubated with exosomes for 1 h and then 100 nM MB was added. As a result, however, there was no significant decrease in the intensity of the fluorescence, even though a high concentration of blocking DNA was used, meaning that almost none of the miRNAs were released out of the exosomes (Fig. 5B). Thus, it is obvious that MB enters the exosomes and hybridizes with the exosomal target miRNAs with high specificity and sensitivity.

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Fig. 6. Detection of miR-21 using MBs in intact exosomes prepared by ultracentrifugation. Exosomes were produced from MCF-7 cells and purified by ultracentrifugation as described in the Materials and Methods section. The indicated amounts of exosomes were incubated with 100 nM MB for 1 h for the detection of exosomal miR-21. Background signal indicates the fluorescence intensity from MB without exosomes, and S/B ratio of each reaction was normalized to control (without exosomes). All values are mean ± SD (**p < 0.01; n ¼ 3).

Fig. 5. Investigation of hybridization of exosomal miR-21 and MB using blocking DNA. (A) 500 nM of blocking DNA was incubated with 100 nM of synthetic miR-21 in the solution for 1 h and then 100 nM of MB was subsequently added and incubated for an additional hour for hybridization. The hybridization in the absence of blocking DNA was used for the comparison. The S/B ratio was significantly decreased by incubation with blocking DNA. (B) 3  1012 exosomes were prepared and incubated with 500 nM of blocking DNA for 1 h. And then 100 nM MB was added to see if the signals from MB and exosomal miR-21 originated from inside or outside the exosomes. All values are mean ± SD (**p < 0.01; n ¼ 3).

3.5. Direct detection of exosomal miR-21 isolated by ultracentrifugation Exosomes from MCF-7 cells also were prepared by ultracentrifugation and incubated with MBs for the detection of miR-21 to verify whether this method is applicable independent of exosome isolation methods. Different concentrations of exosomes were used for the detection, and the signals increased in proportion to the exosome concentration, as shown in Fig. 6. This indicated that direct detection of exosomal miR-21 using MBs can be achieved irrespective of the method used to isolate the exosomes. It was interesting to observe that the intensity of the fluorescence from MB and miR-21 hybridization was higher when using the exosomes that were isolated by ultracentrifugation than it was when exosomes from the Total Exosome Isolation™ process were used. This can be explained in that the quality and/or purity of the isolated exosomes were superior when ultracentrifugation was used instead of the precipitation method. 3.6. Enhanced delivery of MB into exosomes using SLO Streptolysin O (SLO) is a pore-forming bacterial toxin that has been used as a reversible membrane permeabilization tool for live cell imaging of mRNAs using MBs [39,41]. This method is more useful than other delivery methods, including electroporation and microinjection, because it provides non-toxic, simple, and rapid

delivery. It binds to cholesterol on the plasma membrane of the cells and oligomerizes to form pores that are less than 30 nm in diameter, allowing the influx of MBs. It has been reported that exosomes are enriched in raft-associated lipids, such as cholesterol [7]. Thus, we assumed that SLO also can make pores on the surface of exosomes and facilitate the delivery of MB into the exosomes, as shown in Fig. 7A. The same amounts of exosomes were incubated with MBs with and without SLO for 1 h. Different concentrations of SLO, ranging from 0 to 0.8 U/mL, were used to assess the effect of SLO on MB delivery into exosomes. As shown in Fig. 7B, fluorescence signals from MB and miR-21 hybridization in exosomes were increased significantly in the presence of SLO. To rule out the possibility that MB and miR-21 hybridization occurs outside of exosomes since SLO may induce the release of miR-21 from exosome, blocking DNA experiment was carried out. Exosomes were pre-incubated with 0.2 U/mL of SLO for 1 h in the absence or presence of 500 nM of blocking DNA, and then 100 nM MB was added subsequently. As a result, however, there was no significant difference in the intensity of the fluorescence, meaning that miR-21 did not escape exosome during SLO treatment (Fig. S3). As a conclusion, SLO can bind to and make pores on the exosome membrane, thereby enhancing the delivery of MBs and resulting in increased hybridization. Permeabilization of exosomes with 0.1 U/ mL of SLO led to a 137% increase, while 0.2 U/mL of SLO produced an increase of 175% in MB and miR-21 hybridization. Additional increases of SLO up to 0.8 U/mL did not increase the S/B ratio. 3.7. Effect of MB concentration on exosomal miR-21 detection For the most part, we used 100 nM MB for the direct detection of miR-21 in exosomes in this research. Previous reports have indicated that concentrations of MBs of 1 mM or more were used for the detection of mRNAs in live cells [34,35,37]. Increasing the concentration of MB can induce faster hybridization kinetics because more MBs can be delivered into the cells. On the contrary, an increased background signal might interfere with the fluorescence signals from hybridization. Therefore, the MB concentration should be determined considering, for example, the cell type, target mRNA, copy number of targets, and the delivery efficiency. We incubated exosomes with 100 nM or 1 mM MB in the absence or presence of 0.2 U/mL of SLO and observed the changes in the fluorescence intensities at 1 and 2 h after mixing. First, as shown in Fig. 7C, the S/B ratio almost doubled at 1 h after the hybridization reaction began

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Fig. 7. Penetration of MBs into exosomes using streptolysin O (SLO) treatment. Exosomes (3  1010 particles/mL) were incubated with 100 nM MB in the absence or presence of SLO. (A) It is presumed that SLO binds to exosomal cholesterol and makes pores for MB entry into exosomes. (B) Different concentrations of SLO from 0 to 0.8 U/mL were used and the hybridization reactions between MB and exosomal miR-21 were run for 2 h before analysis. Each of the reactions was normalized to the control (without exosomes) in the absence of SLO. (C, D) Different concentrations (100 nM and 1 mM) of MBs were used for the exosomal miR-21 detection in the absence or presence of 0.2 U/mL SLO. Hybridization reactions were run for 1 (C) or 2 h (D), respectively. All values are mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001; n ¼ 3).

by increasing the MB concentration to 1 mM without SLO, meaning that there were more MBs delivered into the exosomes, resulting in a faster hybridization reaction. When the incubation period was 2 h, the fluorescence intensity associated with 100 nM MB increased, but that with 1 mM did not (Fig. 7D). Permeabilization of the exosomes using SLO increased the S/B ratio, and the increment of MB concentration to 1 mM significantly increased the S/B ratio from 25 to 45 in the presence of SLO. Overall, the hybridization of MB and miR-21 soared by more than 400% by combining SLO and the higher concentration of MB within 1 h. The additional increase of S/B ratio from 1 mM MB in the presence of SLO during 1 and 2 h of incubation was relatively small. This probably was due to the saturation in the hybridization reaction by a shortage of MB or the target miRNAs in the exosomes. For the detection of miR-21 from the low amount of exosomes, we have additionally tested lower concentration of MB to decrease the background fluorescence. Five nM of MB were incubated with 0.01 or 0.05  1010 exosomes/mL for 1 h and S/B ratio was measured. Interestingly, as shown in Fig. S4, there was slight increase in S/B ratio. Considering the S/B ratio and exosome concentrations used in Figs. 4A and 7C, this result indicates that the sensitivity can be improved by using appropriate concentrations of MB and exosomes. More detailed experiments using various combinations of MB and exosome concentrations should be followed. 3.8. Detection of exosomal miR-21 in heterogeneous exosomes We showed above that MB can detect miR-21 in exosomes from MCF-7 cells. In contrast, fluorescence signals from CHO-K1 exosomes were very low because CHO-K1 exosomes have low levels of miR-21s compared to MCF-7 exosomes, which was elucidated by the real-time PCR results (Fig. 3). Herein, we prepared a

heterogeneous mixture of exosomes with pre-quantified exosomes from MCF-7 and CHO-K1 cells with a 1:10 ratio in exosome numbers to determine whether miR-21-targeting MB can still detect miR-21 that exists mostly in MCF-7 exosomes. As shown in Fig. 8A, signals from the miR-21 and MB hybridization were much higher from MCF-7 exosomes, even though the number of exosomes was only 10% of the number from CHO-K1 exosomes. And when MB targeting miR-21 was incubated with the mixture of exosomes from both cells, a high S/B ratio was acquired, and it was almost at the level of that from the MCF-7 exosomes added with that from the CHO-K1 exosomes. This was somewhat expected because the amount of miR-21 in MCF-7 exosomes was more than 200 times greater than that in CHO-K1 exosomes, as analyzed with real-time PCR (Fig. 3D). Thus, the fact that the biomarker miRNA level, such as miR-21, was significantly higher in diseased cells than that in exosomes from normal cells provides great opportunities for the diagnosis of disease by exosomal miRNAs. 3.9. Detection of exosomal miR-21 in human serum Serum contains many components that may severely interfere with miRNA detection in the exosomes. Thus, those components must be removed from serum before diagnosis, and this imposes additional resources and processes on the method. Since the ultimate goal of the method is to detect exosomal miR-21 from human samples, including blood, we spiked the exosomes from MCF-7 cells into human serum to see if MB is capable of detecting exosomal miRNAs in human serum without any pre-treatment of the serum. To make sure that the serum used in the experiment has low level of miR-21 probably due to the loss by damage during preservation and distribution, we have measured exosome concentration and relative miR-21 level in serum by ELISA and real-time PCR

J.H. Lee et al. / Biomaterials 54 (2015) 116e125

Fig. 8. Detection of exosomal miR-21 in heterogeneous mixture of exosomes or human serum. (A) Exosomes produced from breast cancer cell line MCF-7 were mixed with higher amount of exosomes from non-cancer cell line CHO-K1 to see if MB still can detect cancer cell-derived exosomes. Single or both of 3  1010 particles/mL of exosomes from MCF-7 cells and 3  1011 particles/mL of exosomes from CHO-K1 cells were incubated with 100 nM MB for the hybridization reaction for 1 h before analysis. (B) 3  1010 particles/mL of exosomes from MCF-7 cells were incubated with 1 mM MB in the absence or presence of human serum. Fluorescence signals were measured 1 h after the hybridization reaction started. All values are mean ± SD (*p < 0.05, **p < 0.01, *** p < 0.001; n ¼ 3e6).

analysis, respectively. The same amount of total RNA (Fig. S5A) or exosomes (Fig. S5B) were used for real-time PCR. As expected, human serum has extremely low level of miR-21 compared to spiked exosomes. Thus, miR-21 from human serum exosomes may not affect the fluorescence detection. One mM MB was added to human serum spiked with 3  1012 exosomes produced from MCF-7 cells. First, as mentioned above, the human serum did not affect the intensity of the fluorescence without the addition of exosomes (Fig. 8B). And although the signals decreased to 62%, we could still get high fluorescence signals from exosomal miR-21 in the human serum. Thus, this result indicated that the in situ detection of exosomal miRNAs using MB can be accomplished without any additional treatment of the serum. 4. Discussion Exosomes are produced by cells and circulate throughout the human body. One of the reasons for exosome research being highly attractive recently is that the exosomes contain precious information from their mother cells. Secreted exosomes can travel along in the circulatory system and transmit signals using their surface ligands or deliver molecules inside target cells. Since exosomes are

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present in most body fluids, including blood, urine, saliva, and breast milk, it is possible to attain minimally- or non-invasive tools for disease detection. Specific miRNAs have been regarded as good indicators for diagnosis, and the aberrant expression of miRNAs has been identified in many diseases [5]. However, miRNAs are intracellular molecules that reside inside the cells, and it is difficult to assess the expression level of miRNAs from the cells in deep tissues. Circulating miRNAs can be used for the practical application of miRNAs as biomarkers [42]. But miRNAs themselves are fragile and are exposed to nucleases in the body's fluids, resulting in the loss of miRNAs. In addition, since they are highly diluted in the fluid, the detection method must have adequate sensitivity, or additional tools will be required for the enrichment of miRNA in compliance with the low concentration of target miRNA. Thus, in those contexts, exosomal miRNAs are excellent biomarkers for diagnostic purposes. Since miRNAs are inside the exosomes, they are protected from exposure to nucleases and shear stress, and these characteristics make exosomal miRNA a consistent source for disease detection. It is well known that both miRNAs and the serum concentration of exosomes are elevated in cancer patients [23]. However, there are hurdles to be overcome in exosomal miRNA detection in many aspects. Real-time PCR analysis is used mainly for the detection of miRNAs from exosomes. This method requires several steps, including exosome isolation, RNA purification, cDNA synthesis, and PCR for quantification. Thus, the analysis is timeconsuming, expensive, and laborious, which are critical impediments for developing commercial disease diagnosis tools. Direct detection of exosomal miRNA using MB as described in this paper can solve the problems associated with real-time PCR analysis. Within an hour, we can obtain significant signals from target miRNA inside the exosomes without any need to isolate miRNAs or synthesize cDNAs. It is noteworthy that MB can detect specific miRNAs in exosomes even in the presence of human serum. Therefore, this method is suitable for high-throughput analysis of exosomal miRNAs for disease diagnosis of large quantities of human samples. The sensitivity of the detection can be improved even more by changing the backbone chemistry of MB, although it must be proven experimentally. The beacons used in the research were 20 -deoxy (DNA) oligonucleotide, and the target was RNA. It has been reported that 20 -O-methyl oligonucleotide in combination with locked nucleic acid (LNA) exhibited higher affinities for RNA, faster hybridization kinetics, and better nuclease resistance [43]. This is due to the fact that those oligonucleotides form an RNA-like 30 -endo conformation, giving a higher melting temperature with the target RNA. In vitro kinetic measurements of chimeric MB that consisted of 20 -O-methyl loop and a 20 -deoxy stem have shown that this structure has the best ability among 20 -O-methyl MB and DNA MB in hybridization with a short RNA target [35]. Hence, direct detection of exosomal miRNA using MB can be developed for enhanced performance by modifying the chemistry of the backbone. For the in situ detection of miRNAs inside the exosomes, it is necessary to deliver MBs into exosomes with no damage. We have shown that, by incubating MB with exosomes, we were able to observe intra-exosomal signals from miR-21 and MB hybridization, meaning that MB had been delivered into the exosomes. It is still unknown how MB can enter exosomes without any treatment. Although it is yet to be investigated, exosomes, unlike cells, may not have active transport systems that can uptake MBs, such as the endocytosis pathways that include clathrin-mediated endocytosis and micropinocytosis. From our experiences with oligonucleotide probes with a fluorescent dye, there is a possibility that a cationic dye, such as Cy3, can penetrate the plasma membrane of cells without any treatment. Even when conjugated with oligonucleotide, Cy3-labeled MB still can enter the cells. Therefore, it is plausible that positively-charged Cy3 allows MB to penetrate into the

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exosomes. Instead of using Cy3 as a fluorescent dye, we synthesized 6-FAM labeled MBs targeting miR-21. The 6-FAM is negatively charged due to its carboxylate anion, but 6-FAM-labeled MBs also entered the exosomes and hybridized with its target miRNA (unpublished result). Thus, how MB enters exosomes remains to be elucidated. Correlation of miR-21 concentrations in solution assay (Fig. 2) versus those in direct detection in exosomes (Fig. 4) can be made. We assume and speculate that each exosome has one miRNA inside, and then 3e6  1010 exosomes/mL which were the concentrations of exosomes used in Fig. 4A equal to 3e6  1010 miR-21/mL. Using Avogadro number (6.02  1023), the concentrations of miR-21 will be 50e100 nM which are the concentrations for comparable S/B ratio in solution assays shown in Fig. 2B. Theoretically, moreover, 100 nM MB equals to 6.02  1010 MB molecules/mL, which is slightly higher than the concentration of exosomes (3e6  1010 exosomes/mL for example). Considering the delivery efficiency of MB and number of miR-21 in one exosome, it is likely that we need to have more MBs for each exosome to have at least one MB. This can be supported by the result shown in Fig. 7C that increasing MB concentration to 1 mM or the permeabilization by SLO almost doubled S/B ratio, respectively. For miR-21 detection in exosomes isolated by ultracentrifugation, on the contrary, 100 nM MB provides sufficient amount of MB to exosomes (5e10  107 exosomes/mL for example) as shown in Fig. 6. Therefore, we suggest that the optimal concentration of MB in exosomal miRNA detection should be determined based on exosome concentration, target miRNA concentration in exosome, miRNA sequence, isolation method, delivery method, etc. Developing a method for the acceleration of MB delivery into exosomes can be accomplished by considering previous methods that have proven effective in cellular oligonucleotide delivery. For instance, electroporation of exosomes with MB may efficiently delivery MBs, but electrical shock may affect the exosomes' integrity, and additional equipment and materials for electroporation are required, causing the detection process to be more difficult and impractical in many aspects. Microinjection is not applicable to exosome research because the exosomes are too small, and the method itself is labor-intensive and only practical with a small number of cells. Cell penetrating peptide-linked (CPP-linked) MB was developed for detecting RNA in live cells [44]. Depending on the conjugation peptide and chemistry, it is possible to deliver MBs into the cytosol or the nucleus. However, this does not necessarily mean that the method also can work with exosomal MB delivery, because exosomes do not have all the components that cells have. Moreover, some reports have suggested that CPP-induced cargo internalization is mainly due to endocytosis, although this still is not clear [44,45]. If this is the case, we cannot expect to use CPPconjugated MBs for the detection of exosomal miRNA. In this report, the permeabilization of exosomes with SLO successfully enhanced the delivery of MBs. As described above, exosomal cholesterols provide opportunities for the binding and polymerization of SLO on the exosome membrane, thereby forming pores for MB entry. Increased MB delivery gave rise to a higher opportunity for hybridization between MB and miR-21, which may have been due to the increased concentration of MB in the exosomes. Further increases in the SLO concentration did not yield higher hybridization signals, probably because of exhaustion of the cholesterols or the limitation of target miR-21s. The latter may not be the case here, because increasing the concentration of MB to 1 mM resulted in increased hybridization, as shown in Fig. 7C and D. thus, it can be concluded that the direct detection of exosomal miRNA using MB can be improved by permeabilization with SLO. The technique for detecting miRNAs in the intact exosome using MB shown here can be potentiated more by combining it with an

exosome capture technique. This removes a step for exosome isolation, thereby shortening the overall time required for analysis. Moreover, commercially available methods for exosome isolation involve the precipitation of exosomes, which also results in the precipitation of unwanted impurities from the cells. They also require overnight incubation to prepare the exosomes. Ultracentrifugation may solve this problem, but considerable time is still required to isolate the exosomes, and, practically, only a small number of samples can be processed at the same time due to limitation of the ultracentrifuge itself. Besides, the exosomes from diseased cells might be diluted in the circulatory system. This is a critical problem for the most of miRNA detection technique including PCR-based method that all the exosomes have to be lysed together regardless of their origins for the miRNA extraction. As a result, biomarker miRNAs in exosomes from diseased cells are mixed and diluted with those from healthy cells. For in situ detection of miRNA in whole exosomes using MB, this problem can be overcome by capturing and separating exosomes originated from diseased cells from serum using antibody that recognizes exosome surface disease marker. Therefore, developing a method for exosome enrichment from samples together with the direct miRNA detection method using MBs will synergistically accelerate the development novel methods for the diagnosis of diseases. Recently, for instance, Kanwar et al. reported a microfluidic device, called ExoChip, for the isolation of circulating exosomes on polydimethylsiloxane (PDMS) chips by conjugating CD63 antibodies [46]. After capturing exosomes, they extracted exosomal RNAs on the chip and executed several experimental steps for miRNA analysis. In our future studies, we will assess the combination of the method introduced in this study using MBs for the direct detection of miRNA in the exosomes with a microfluidic platform for the fast, simple, easy, and sensitive detection of exosomal miRNAs with reduced labor and cost. 5. Conclusion We showed that exosomal miRNA can be detected directly using miRNA targeting oligonucleotide probe, i.e., the molecular beacon. As a model system, we used the breast cancer cell line, MCF-7, for exosome production because it has a high level of miR-21 as a target miRNA. We successfully demonstrated that MB can visualize target miR-21 in the cancer cell-derived exosomes, even in the presence of human serum. The method can be used irrespective of the exosome isolation method used, including ultracentrifugation, ExoQuick-TC™, and Total Exosome Isolation™. In addition, exosome permeabilization with SLO could enhance the entry of MBs into exosomes and the hybridization signals as a result. The method also can discriminate specific exosomes produced from cancer cells even when mixed with exosomes from other types of cells with low levels of target miRNA. The method shows significant promise for use in detecting miRNAs from exosomes in human serum. The direct detection technique of exosomal miRNA using MB will open great opportunities for cancer diagnosis, prognosis, and response to treatment. Assuming that the proposed method can be combined with point-of-care testing high-throughput diagnostic equipment, the detection of miRNAs in exosomes allows for feasible system that can be used for the fast and easy pre-clinical diagnosis of various diseases. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (NRF2012R1A1A1040652) and Ministry of Education (NRF-

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