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Rapid Capture and Non-destructive Release of Extracellular Vesicles using Aptamer-based Magnetic Isolation Kaixiang Zhang, Yale Yue, Sixuan Wu, Wei Liu, Jinjin Shi, and Zhenzhong Zhang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00060 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Rapid Capture and Non-destructive Release of Extracellular Vesicles using Aptamer-based Magnetic Isolation Kaixiang Zhang, †, a, c, d Yale Yue, †, a, b Sixuan Wu, a, c, d Wei Liu, a, c, d, * Jinjin Shi, a, c, d, * Zhenzhong Zhang a, c, d, * a.

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China.

b.

Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450001, China.

c.

Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China.

d.

Key laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, China.

†

K. Zhang and Y. Yue contributed equally.

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

ABSTRACT Extracellular vesicles (EVs) play important roles in cell-cell communication by transferring cargo proteins and nucleic acids between cells. Due to the small size (50-150 nm) and low density, rapid capture and nondestructive release of EVs remains a technical challenge, which significantly hinder its bio-function study and biomedical application. To address this issue, we designed a DNA aptamer-based system that enable rapid capture and non-destructive release of EVs in 90 mins, with similar isolation efficiency to ultracentrifugation (around 78%). Moreover, since we designed a DNA structure-switch process to release the exosomes, the isolated EVs maintained high bioactivity in cell-uptake assay and wound-healing assays. Using this method, we can isolate EVs from clinical samples and found that the amount of MUC1 positive EVs in breast cancer patient plasma sample is significantly higher than healthy donors. This DNA aptamerbased magnetic isolation (AMI) strategy can be potentially applied for the biofunction study of EVs and EVbased point-of-care clinical test. Keywords: Extracellular vesicles, DNA aptamer, magnetic isolation, DNA structure-switch, non-destructive release.

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Extracellular vesicles (EVs), including exosomes and microvesicles, are cell derived phospholipid vesicles, actively secreted by a variety of cells.[1-3] EVs play important roles in mediating cell-cell communication by encapsulating and delivering proteins, mRNA, miRNA and DNA between cells.[4-7] Although the bioactivity of EVs has not been fully understood, growing evidences are showing that EVs can regulate tumor microenvironment[8], prepare pre-metastatic niche[9] and contribute to chemotherapeutic resistance[10]. Besides, high concentration of EVs can be found in many bodily fluids, such as saliva and blood[11,12]. Therefore, EVs can be potentially used as targets for tumor therapy as a non-invasive diagnostic and prognostic biomarker. The isolation and enrichment steps are necessary preanalytical procedures for investigating the function of EVs, as well as application in clinical translations.[13,14] A variety of EVs enrichment or isolation methods have been developed, including ultracentrifugation[15], density gradient separation[16], size exclusion chromatography[17] and polymer-based precipitation[18]. However, these methods are usually time-consuming and may lead to impurities and damage of EVs[19]. Therefore, some new microfluidics systems[20-23] or magnetic isolation methods[24] have also been developed to improve EVs enrichment efficiency and to make the isolation process faster and less complex. However, most of the developed system are still using antibodybased separation, which is difficult for achieving non-destructive release of EVs and may affect the flowing biofunction study and clinical diagnostic applications. Recently, some lipid-based nanoprobes[24], anion exchange (AE)-based isolation[25], and cholesterol-modified magnetic beads[26] have been developed for EVs isolation and achieving high reproducibility and high purity, which can be applied for plasma sample analysis. In this work, we think about using another kinds of binding ligand: DNA aptamer for EVs isolation. Test the capture efficiency of CD63 aptamer and whether the structure-switch can release EVs from capturation. DNA aptamers are well-developed binding agents for various targets, including proteins, small molecules 2 ACS Paragon Plus Environment

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and cells[27-34]. Some aptamer-based exosomes analysis methods have been developed for colorimetric profiling of exosomal proteins, which identified various kinds of aptamers that can bind to EVs.[35-37] Besides, the DNA aptamer structure can be easily disrupted by complementary sequences or cleaved by restriction enzymes, which permits the captured EVs to be nondestructively released from the binding surface for further biofunction study or molecular analysis.[38-40] Recently, Liu et al. used λ-DNA and DNA aptamer for sorting and analysis of EVs and achieved simultaneous size-selective separation and surface protein analysis of individual EVs.[41] Dong et al. reported an aptamer-magnetic bead bioconjugates to capture tumor exosomes and analyze the released messenger DNAs using electrochemical signals.[42] Xu et al. presented a microfluidic platform for EVs isolation and in situ electrochemical analysis using DNA aptamer sensors.[37] However, according to our knowledge, a systematical research of using DNA aptamer-based magnetic isolation to achieve highly efficient and non-destructive EVs separation, and characterizing the morphology and biological functions of isolated EVs has not been thoroughly studied. Herein, we reported a DNA aptamer-based magnetic isolation system (AMI) for rapid capture and nondestructive release of EVs. Using an aptamer to bind CD63 protein which is overexpressed on the surface of EVs, we can capture and isolate EVs from cell culture media and plasma, meanwhile maintaining a high purity of the captured EVs. Importantly, by adding complementary sequences to break the secondary structure of aptamers, we can release the captured EVs from magnetic beads without any damage to EVs and the whole process takes less than 90 mins.

RESULTS The mechanism of aptamer-based EVs isolation process was illustrated in Figure 1. Cell culture media or blood samples were first briefly centrifugated to exclude cell debris and red blood cell. Since EVs are known to express high level of transmembrane protein CD63[24]. Biotin modified CD63 aptamer was used to mark 3 ACS Paragon Plus Environment

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EVs in the solution. Afterwards, the streptavidin modified 500 nm diameter magnetic beads were added to capture the aptamer marked EVs, and the EVs can be captured on the surface of beads and magnetically separated from the supernatant. After 3 times washing with PBS, the complementary oligonucleotide was added to hybridize with CD63 aptamer and break the aptamer structure to release EVs from the magnetic beads. The magnetic beads before and after capture of EVs were first characterized by TEM and SEM. As shown in figure 2a, 2b, 2c and 2d, the monodispersed magnetic beads were with a mean size of around 500 nm. After capturing EVs, obvious spherical shape vesicles can be seen on the surface of magnetic beads (Figure 2b, 2d) and each bead can capture multiple EVs. These imaging data clearly showed that the CD63 labelled EVs can be captured on the surface of streptavidin modified magnetic beads. GAPDH is regarded as cellular internal reference protein and has low expression level in EVs. In this experiment, we use it to reflect the amount of protein loading and as a negative marker for EVs[24]. CD63 is a tetraspanin protein which has been shown to participate in endosomal vesicle trafficking and usually used as common EV markers. Therefore, we used Western Blot to analyze GAPDH and CD63 expression from MCF-7 cell lysates and EVs protein lysates, respectively. As expected, the CD63 expression level of EVs was significantly higher than cells, which indicated that our method can efficiently isolate EVs from cells (Figure 2e). Afterwards, the isolated EVs were characterized and quantified using nanoparticle tracking analysis (NTA) system. As shown in figure 2f, the isolated EVs were with uniform size distribution and the diameter was around 80 nm. After the capture process, the concentration of EVs in standard sample was significantly decreased, and the concentration of nEvs before and after capturing were measured by NTA (Figure S1). Result shows that isolation efficiency with optimized conditions was around 78%. The data are shown in Table S1-S4. 4 ACS Paragon Plus Environment

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A comparison between the EVs purified by ultracentrifugation and aptamer-based magnetic isolation was conducted using TEM and NTA analysis. The pictures of TEM show that EVs isolated by AMI are more uniform than those enriched by standard ultracentrifugation methods (Figure 3a, 3b). This may because the ultracentrifugation process may damage the EVs structure due to the extremely high forces, and some bulky protein could also be in the sedimentation which can significantly affect EVs purity. Comparing with the EVs enriched by ultracentrifugation, NTA analysis indicates that EVs isolated using AMI have much narrower size distribution (Figure 3c, 3d). We assume this is because the nucleic acid hybridization process enabled non-destructive release of EVs, which can better maintain the morphology and membrane protein of EVs. To test the capture specificity, the non-specific adsorption of MBs before and after blocking was firstly investigated (Figure4a). According to NTA test, without blocking, the MBs itself will absorb around 22% of total exosomes. After blocking with BSA, the ratio was reduced to 2.5%. This result indicates that most nonspecific absorption of MBs can be avoided by the BSA blocking processing and all the subsequent experiments were carried out using MBs after blocking. The specificity of CD63 aptamer was also tested (Figure 4b, 4c). Specifically, the CD63 aptamer and random sequences were modified on glass slides and the fluorescent labelled exosomes were incubated with the two kinds of surface and then imaged using confocal microscopy. According to the fluorescent image (Figure 4b) and exosomes number quantification (Figure 4c), the amount of exosomes captured by CD63 aptamer is significantly higher than random sequence and PBS group, indicating CD63 aptamer has good capture efficiency and specificity. To achieve high EVs isolation efficiency, different factors that may affect the isolation efficiency of AMI have been carefully tested. The amount of aptamer was firstly checked (Figure 5a, Table S1) and the isolation 5 ACS Paragon Plus Environment

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efficiency was found to increase gradually with the increasing amounts of CD63 aptamer and reach a plateau at 10 nM. An additional quantity of DNA aptamer did not further increase the isolation efficiency. Similarly, the aptamer incubation time, magnetic beads concentration and magnetic beads incubation time were subsequently tested (Figure 5b, 5c, 5d; TableS2, S3, S4). The incubation time optimization data showed that 15 min for each step is adequate and prolonged incubation did not provide any statistically significant benefit. In the optimized condition, 100 μL 2×108 particles/ml streptavidin modified MBs can bind to about 1 pmol CD63 aptamer. Therefore, each magnetic bead was estimated to bind about 3 x 104 aptamer molecules. After optimization, the total AMI process takes less than 90 mins (including the magnetic isolation and washing step). Moreover, the whole AMI process does not need time-consuming multistep procedures and expensive apparatus. Comparing with the conventional ultracentrifugation methods which usually take more than 6 hours and complex ultracentrifugation process, this aptamer-based magnetic isolation process is much faster and can be performed with routine sample processing steps. After magnetic isolation, the complementary DNA sequences were added to break the secondary structure of aptamer and release EVs from magnetic beads. As illustrated in figure 4e, 4f, the release efficiency is around 78% with the addition of 1.5 μM complementary DNA sequences (Table S6). Besides, the rapid capture and non-destructive release feature of AMI showed the promise for fast collection of specific subtypes of EVs by targeting known surface proteins. Moreover, these high capture and release efficiency allow us to do further tests to study the bioactivity of isolated EVs. To evaluate the bioactivity of EVs isolated by AMI, we first tested whether the isolated EVs can be absorbed by cells with a cell-uptake assay (Figure S2). Specifically, fresh cell-culture mediums of MCF-7 cells were collected and incubated with phospholipid membrane staining dye (5 μM Dil) for 10 mins at room temperature. Afterward, EVs were isolated by AMI and the released fluorescent labelled EVs were then 6 ACS Paragon Plus Environment

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incubated with MCF-7 cells for different time. The fluorescence images and the quantitative data show that the Dil labelled EVs can be quickly uptaken by MCF-7 cells. The result indicates that the AMI isolated EVs may maintained a degree of homology. It has been reported that EVs can mediate stromal mobilization of autocrine Wnt-PCP signaling and promote cell migration[43,44]. To test whether AMI isolated EVs maintained this biofunction, a wound healing assay was conducted to evaluate the ability of AMI isolated EVs for promoting cell migration (Figure 6). The cell migration was assessed at 12 h and 24 h time points after wounding. As shown in Figure 5b and 5c, the quantification of wound closure indicated that the AMI isolated EVs can induce significantly more migration comparing to the blank control, which confirmed that the AMI isolated EVs maintained their biofunctionality to promote cell migration. Finally, to test the potential of AMI for clinical application, we collected 8 plasma samples from 4 different healthy donors and 4 breast cancer patients with permission. A repetitive test was firstly performed to evaluate the reproducibility of AMI. As shown in Figure S3, AMI methods can stably capture around 60% EVs from the plasma sample and release around 20%. The result shows that AMI for isolating EVs from plasma samples was with lower efficiency than from cell-culture medium, which may due to the influence of complex plasma environment on nucleic acid hybridization. CD63 aptamer based magnetic isolation was then used to isolate EVs from the plasma samples of 4 healthy donors and 4 breast cancer patients. The isolation efficiency was measured by NTA. As shown in Figure 7a, Table S7 and Table S8, there is no significant difference in the amount of isolated EVs between healthy donor and cancer patients, which may because CD63 is a broad marker of EVs without tumor specificity. Therefore, we then used a tumor biomarker MUC1 aptamer to specifically capture tumor-associated EVs. As shown in Figure 7b, Table S9 and Table S10, there is significant difference in the amount of isolated EVs between 7 ACS Paragon Plus Environment

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healthy donor and cancer patients using MUC1 aptamer, which show high potential of AMI for diagnostic and prognostic application.

Discussion In summary, we developed a DNA aptamer-based magnetic isolation method for rapid capture and nondestructive release of EVs from cell culture medium and human plasma sample. Using DNA aptamer as a recognition and capture agent, we developed this AMI method to achieve EVs with high recovery efficiency (78%) and less protein impurities in less than 90 mins. Comparing with previously developed EVs isolation process, we can use complementary DNA sequences to release captured EVs from magnetic beads without any disturbing to the EVs structure or surface protein markers. The bioactivity of isolated EVs has been tested using cell-uptake assay and wound healing assay, which showed high potential for downstream biofunction studies. Besides, AMI has been used for isolating EVs from human plasma sample and showed good reproducibility. This rapid and nondestructive EVs isolation methods can be potentially applied for studying the biofunction of EVs or profiling the surface marker of EVs for clinical translation of diagnosis and prognosis.

Methods Materials and Apparatus. All synthetic DNA oligonucleotides were purchased from Sangon Biotech (Shanghai China). Streptavidin-modified magnetic beads were purchased from Thermo Fisher Scientific (Waltham, MMAS, USA). Centrifugal filter units were purchased from Millipore (MMAS, USA). Rabbit CD63 antibody was from Abcam (Cambridge, UK). Rabbit anti-GAPDH antibody was purchased from Hangzhou Goodhere Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was purchased from Proteintech (Chicago, USA). Beckman Coulter OptimaTM XPN ultra-centrifuge and Thermo Fisher Scientific refrigerating centrifuge 8 ACS Paragon Plus Environment

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was used for centrifugation. Malvern Nanosight NS300 was used for EVs concentration analysis. FEI Tecnai transmission electron microscope was used for TEM imaging. Hitachi cold filed emission scanning electron microscope was used for SEM imaging. Leica TCS SP8 laser scanning confocal microscopy was used for taking fluorescent images. Cell culture. MCF-7 cells were cultured in Solarbio RPMI 1640 with 10% (v/v) fetal bovine serum. Cells were maintained in 5% CO2 and 95% air at 37 °C in a humidified incubator. For EVs collection, MCF-7 cells were cultured in cell culture flasks for two or three days until they reached 80%. Then, cells were washed three times with PBS and cultured in serum free medium for 48 h and the serum free medium was collected for ultracentrifugation based EVs isolation. Ultracentrifugation based EVs isolation. The serum free cell culture medium was collected and centrifuged at 1,000 x g for 5 minutes to remove the cells, and the supernatant was centrifuged at 16,500 x g for 20 minutes to remove large vesicles in solution. followed by a filtration step using 0.22 μm (pore size) filters to remove cell debris. Then the cell culture medium was ultracentrifuged at 100,000 x g for 1 hour at 4 °C to isolated the EVs. The stock solution and the standard EVs samples were stored at -80 °C before use. Characterization of EVs. TEM, SEM, Western blot and NTA measurement were used for characterizing the isolated EVs. For TEM imaging, 10 μL isolated EVs sample was dropped on to a 200-mesh copper grid and incubated for 2 minutes at room temperature and the excess sample was removed. Uranyl acetate was used to negatively stain the EVs for 1 minute. The samples were examined with FEI Tecnai transmission electron microscope and the accelerating voltage was 100kV. For SEM imaging, 5 μL magnetic beads capturing EVs were dropped onto a polished silicon wafer. The images of MBs and MBs covered with EVs were acquired with Hitachi cold filed emission scanning electron microscope. 9 ACS Paragon Plus Environment

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The concentration of EVs in different sample were measured using Malvern Nanosight NS300. The isolated EVs were diluted 100 times, injected into the chamber and analysed using NTA software to count the number of EVs. The NTA measurement movie was in the supporting information. Preparation of plasma samples. Healthy human blood samples were obtained from Hospital of Zhengzhou University with permission. Plasma samples were collected by centrifuging blood at 3,000 x g for 10 minutes at 4 °C. Then the plasma samples were filtered with a 0.22 μm filter and stored at -80 °C before use. Estimation of the non-specific adsorption of MBs. Streptavidin modified MBs was added into 500 μL blocking buffer (10 mM pH 7.4 Tris-HCl, 1 mM EDTA, 2 M NaCl, 0.01% Tween-20, 0.05% BSA), and slowly shake at room temperature for 1 hour (keeping the MBs in a dispersed state). Then the MBs were separated by magnet and washed three times with PBS containing 0.01% Tween-20. The non- specific adsorption efficiency were calculated by measuring the concentrations of EVs before and after absorption using NTA. Specificity of CD63 aptamer. The CD63 aptamers(5’-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTT TTT TTT TTT TTT - 3’-Biotin) and random sequence (5’- TGT GCG GCG AAA TAT TAT AGC TAC CGC AAT TAC TTT TTT TTT TTT TTT - 3’ -Biotin) were synthesized in Shanghai Sangon Co., Ltd. and purified by HPLC. The experiment was conducted in small wells with a diameter of 0.5 cm and the bottom of each well was streptavidin-modified. The exosomes form MCF-7 cells were collected and incubated with 5 μM Dil for 10 mins at room temperature. The concentration of MCF-7 exosomes were counted to be 1.4×107 particles/ml using NTA. The collected exosomes were separated to two groups and incubated with CD63 aptamer and random sequence, respectively,, then washed with PBS for 3 times. Then, the sample were imaged using confocal microscopy. The amount of exosomes captured by CD63 aptamer and random sequence were counted by 10 ACS Paragon Plus Environment

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fluorescent image to evaluate the specificity of CD63 aptamer. Aptamer-based EVs isolation. CD63 aptamer (5’-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTT TTT TTT TTT TTT - 3’-Biotin) and MUC1 aptamer (5'- Biotin- TTTTTTTTTT GCA GTT GAT CCT TTG GAT ACC CTG G -3') were purchased from Sangon Biotech. EVs capture steps were as follows: First, Biotin labelled CD63 aptamers were used to identify and bind to EVs in 500 μL sample. Then, the streptavidin-conjugated magnetic beads (MBs) (Invitrogen) were added to capture the aptamer labelled EVs. For capture optimization, we studied the effect of aptamer concentrations, incubation time and MBs concentration by evaluating the capture efficiency. Capture efficiency was measured by NTA. Release of captured EVs. Complementary DNA sequence (5’- GTG GGG TGG AGC GAG GGC ACT GTG ATT ACG ATA AAA AAA AAA AAA AAA-3’) and (5'- CCA GGG TAT CCA AAG GAT CAA CTG CAA AAA AAA AA -3') was used to open aptamer structure and release EVs from MBs. The EVs release processes are as follows: MBs with EVs was washed 3 times by PBS and then redistributed in 500 μL PBS. Complementary DNA sequences were then added to the redistributed samples and incubated at 37 °C for 15 min. After magnetic separation, the release efficiency was calculated by measuring EVs concentration of the supernatant using NTA. The isolation efficiency = The amount of released EVs / the amount of total EVs ×100%. Cellular uptake assay. 500 μL model EV sample was incubated with 0.01 mg/mL Dil (Beyotime Biotechnology) for 10 mins at room temperature and then isolated using AMI. All the sample processing steps were operated in darkness. MCF-7 cells were cultured in a 6-well plate in 5% CO2 at 37 °C. When the cell confluence reached 80%, the cell culture medium was removed and cells were stained with Hoechst 33342 (Solarbio) for 5 minutes. Then cells were washed 3 times and then incubated with Dil stained AMI isolated EVs. The cellular uptake assay was imaged with a confocal microscopy in 2 hours, with a time 11 ACS Paragon Plus Environment

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interval of 5 mins. Wound-healing assay. MCF-7 cells were firstly cultured in a 6-well plate overnight. When the cell confluence reached 100%, the cell monolayer was scratched using a pipette tip and the detached cells were removed by replacing the cell culture medium. The scratched cells were treated with 2 ml cell culture medium containing 9.4×107 isolated EVs and cultured at 37 °C in 5% CO2 for 12 h and 24 h. The control experiment was performed the same way using cell culture medium without EVs. The wound width was measured using microscope. Microscopy images and ImageJ were used to calculate the wound areas. Application for clinical samples. 8 plasma samples from 4 different healthy donors and 4 breast cancer patients were collected with permission. 5 μL collected plasma sample was first diluted in 495μl PBS and then incubated with 10 nM CD63 or MUC1 aptamers at 37 °C for 15 minutes. Afterwards, 10 μL 1 mg/mL magnetic beads were added to capture the aptamer labelled EVs for 15 minutes at 37 °C. After magnetic isolation, the capture efficiency was calculated by measuring the concentration of EVs before and after capturing. The magnetic beads with captured EVs were resuspended in 500 μL PBS, which contained 1.5 μM complementary DNA sequence and incubated for 15 mins. The released EVs concentration was measured by NTA to estimate the release efficiency.

Supporting Information Available: The following files are available free of charge. NTA quantification results, Cell-uptake assay.

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ACKNOWLEDGEMENTS The work is supported by grants from the National Natural Science Foundation of China (No. 81601597, U1704178), key scientific research projects (Education Department of Henan Province, No.17A350003), Postdoctoral Science Foundation of China (No. 2015M582210, 2018T110745, 2017M622380) and the Zhengzhou University Initiative Scientific Research Program (No. 32210809). Thanks to the modern analysis and computing center of Zhengzhou University for technical assistance.

AUTHOR CONTRIBUTIONS 17 ACS Paragon Plus Environment

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. K. Zhang and Y. Yue contributed equally.


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Figure 1. Schematic diagram of DNA aptamer-based magnetic isolation (AMI) process for EVs isolation. EVs from either cell culture medium or blood plasma are first marked with biotin labelled CD63 aptamer, then separated using streptavidin modified magnetic beads. After magnetic isolation, the complementary sequences, which can hybridize with the CD63 aptamer to break the aptamer secondary structure, were added for non-destructive release of the EVs from magnetic beads.


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Figure 2. Characterization of size, morphology and protein expression of the isolated EVs from standard sample. a) TEM image of the streptavidin modified magnetic beads. b) TEM image of the magnetic beads capturing EVs on the surface. c) Cryo-SEM image of the streptavidin modified magnetic beads. d) Cryo-SEM image of the magnetic beads capturing EVs on the surface (the spherical vesicles labelled using white arrow). e) GAPDH and CD63 expression from isolated EVs and their corresponding cells were identified using Western Blot. f) NTA analysis of EVs of standard sample.


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Figure 3. Comparison of the morphology, concentration and size distribution of EVs isolated by ultracentrifugation and AMI. a) TEM analysis of EVs isolated by ultracentrifugation. Scale bar: 100 nm. b) TEM analysis of EVs isolated by AMI. Scale bar: 100 nm. c) Analysis of the concentration and size distribution of EVs isolated by ultracentrifugation using NTA. d) Analysis of the concentration and size distribution of EVs isolated by AMI using NTA.


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Figure 4. Investigation of non-specific absorption of MBs and the specificity of CD63 aptamer. a) The EVs adsorption efficiency of MBs before and after BSA blocking. (3 independent experiments, T Test, **p < 0.01). b) Quantification of the EVs captured by CD63 aptamer and random sequence. (3 independent experiments. T Test, ***p < 0.001) c) Confocal microscopy of EVs captured by CD63 aptamer, random sequence. (Scale bar: 10μm).


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Figure 5. Capture and release efficiency of AMI are influenced by a) aptamer concentration (1pM to 15 nM), b) incubation time of aptamer and EVs (1 min to 30 min), c) the concentration of streptavidinconjugated magnetic beads (MBs) (1 x 107 particles/mL to 3 x 108 particles/mL), d) incubation time of MBs and aptamer marked EVs (1 min to 30 min), and e) the concentration of hybridization sequence (10 nM to 2 μM). Error bars are based on triplicate independent experiments. f) The overall optimized EVs capture and release efficiency of AMI.


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Figure 6. Wound-healing assay for MCF-7 cells treated with the isolated EVs. a) The images were taken at 12 h and 24 h after the wounding using inverted microscopy. Scale bar: 500μm. b, c) The wound healing speed is significantly increased by incubation with isolated EVs (3 independent experiments, T Test, **p < 0.01), indicating that the AMI isolated EVs hold strong activity to promote cell migration.


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Figure 7. Isolation of EVs from healthy donor and cancer patient plasma samples. a) CD63 aptamer based magnetic isolation of EVs in 4 healthy donor and 4 breast cancer patient plasma samples. b) MUC1 aptamer based magnetic isolation of EVs in 4 healthy donor and 4 breast cancer patient plasma samples. The isolation efficiency was measured by NTA. Error bar is based on triplicate experiments. p < 0.01 (Student T test).


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