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Ultrasensitive and reversible nanoplatform of urinary exosomes for prostate cancer diagnosis Ping Li, Xiyuan Yu, Wujuan Han, Ying Kong, Weiyang Bao, Jiaqi Zhang, Wancun Zhang, and Yueqing Gu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00621 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019
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Ultrasensitive and reversible nanoplatform of urinary exosomes for prostate cancer diagnosis Ping Li†, §, Xiyuan Yu†, §, Wujuan Han†, Ying Kong ‡, Weiyang Bao†, Jiaqi Zhang†, Wancun Zhang† and Yueqing Gu*, †, ‡ †Department
of Biomedical Engineering, School of Engineering, China Pharmaceutical University, No. 639 Longmian Avenue, Jiangning District, Nanjing 211198, China ‡ State Key Laboratory of Natural Medicine, China Pharmaceutical University, No. 639 Longmian Avenue, Jiangning District, Nanjing 211198, China KEYWORDS urinary exosomes, cancer biomarker, prostate cancer diagnosis, prostate specific membrane antigen, aptamer ABSTRACT: Prostate cancer cell-derived exosomes in urine have been extensively studied recently and regarded as novel biomarkers for cancer diagnosis and prognosis which present wide prospects in clinical applications. Sensitive detection and specific capture methods are essential for exosomes analysis. Herein, a dual functional platform composed of superparamagnetic conjunctions and molecular beacons (SMC-MB) is reported. The SMC-MB platform is designed based on aptamer immunoaffinity with ultra-sensitive detection efficiency and reversible isolation capacity, which respectively profit from nonenzymatic amplification methods and magnetic separation along with restriction cleavage. It was noteworthy that exosomes quantification was exactly amplified and transformed into single strand DNA detection. Correlated measurements evidence that LOD of SMC-MB is as low as ~100 particles /μL in urine, and a linear relationship meets between logarithmic concentration of exosomes and fluorescence intensity of molecular beacon. Furthermore, employing prostate specific membrane antigen (PSMA) aptamer, the platform adapted to detect and capture PMSA-positive exosomes from urine samples provides excellent diagnostic efficiency for prostate cancer (PCa). The expression of typical biomarkers of PCa, i.e., PSA and PCA3 mRNA are significantly higher in PSMApositive exosomes. Altogether, the platform and strategy described in this paper are promising in urinary exosomes analysis and prostate cancer detection.
Exosomes are nanoscale extracellular vesicles generated by inward budding of intercellular endosomes which are in diameters of 30-150nm, and have arrested much attention due to their abundant bio-information alongside the important roles in intracellular communication.1-3 Cancer-derived exosomes have been identified important in cancer development, progression as well as drug resistance. Since cancer related pathogenic components, including messenger RNA (mRNA), microRNA (miRNA), noncoding RNA (ncRNA), DNA, membrane and cytosolic proteins and lipids46can be delivered and exchanged through the double lipid membrane vesicles, thus contributing to the recruitment and reprogramming of the tumor microenvironment.7-8 Accordingly, exosomes are of great significance in cancer diagnosis, as the amount of secreted exosomes together with cancerous nucleic acid and protein they contain allow the prediction of cancer risk.9-10Circulating exosomes in urine, blood and saliva can be non-invasive or minimally invasive biomarkers.11-12 Compared with serum or plasma, urine yields large volumes and permits routinely collection. 13 Urine exosomes have been proposed as treasure chests of biomarkers especially for some tumor located anatomically proximate to the urethra.14 Notably, recent studies have proved that prostate cancer (PCa) derived exosomes are enriched in urine after a
slight prostate massage, which assists the discovery and detection of prostate cancer.15 Even though prostate specific antigen (PSA) has shown reasonable sensitivity for prostate cancer screening, diagnosis as well as prognosis, one of the drawbacks is low specificity, such that benign hyperplasic conditions can also lead to PSA increment.16 Thus, tumorderived exosomes detection is a novel approach to find predictive markers for PCa according to numerous studies. Relatively, prostate specific membrane antigen (PSMA) positive exosomes demonstrate obvious merits in PCa diagnosis, and offer essential biomarkers for the development of biosensor-based PCa detection methods. 17 Although increasing studies have focused on urinary exosomes, it is still challenging to isolate and detect exosomes from complex matrix. To date, conventional methods for exosomes detection including transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), western blot and ELISA which are either time-consuming, unwieldy, inconvenient or expensive.18-19The main techniques for highly efficient separation of exosomes are ultracentrifugation, immunoaffinity capture, exosome precipitation and microfluidics-based methods.20-22 In addition, ultracentrifugation serves as the gold standard for total exosomes, while immunoaffinity method can accurately
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capture exosomes with specific surface proteins, but both of them are expensive, irreversible and
Figure 1. Schematic illustration of SMC-MB platform. (a) Construction of SMC-MB platform. (b) Procedure of SMC-MB platform in exosomes analysis. (c) Principle of nonenzymatic amplification cycle. (d) Exosomes purification by the restriction enzyme. In addition, different from other irreversible immunoaffinity mono-functional in clinical separation.23 Recently, convenient isolation methods, restriction sites were introduced to purify and quick methods with high sensitivity and specificity have target exosomes from the superparamagnetic platform. Finally, been reported. Researchers prefer to isolate exosomes by exosomes were further studied for cancerous properties. magnetic separation along with electrochemical 24-25 and Above all, this platform can be used to isolate and detect PCa microfluidic methods, 26 Surface enhanced Raman scattering related exosomes, revealing application potential in prostate (SERS) 27 probes and fluorescent probes 28 to detect exosomes cancer diagnosis and prognosis. simultaneously. However, limitation of low concentration as well as complex background of urine is non-ignorable in RESULTS AND DISCUSSION exosomes collection and detection. As a result, signal Principle of SMC-MB platform. The capture and amplification and reversible strategy should be further detection mechanism of the SMC-MB platform for PCa developed. related exosomes is exhibited in Figure 1. SMCs were Herein, a dual functional platform composed of comprised of superparamagnetic Fe3O4 cores and specific superparamagnetic conjunction and molecular beacon (SMCaptamer-ssDNA complex (Figure 1a). All sequences applied in MB) was reported to separate and quantify PCa-related this paper were listed in supporting information Table S1. exosomes, and convert exosome detection to single strand After incubated with exosomes samples, two steps were DNA (ssDNA) detection. Typically, PSMA (prostate specific carried out to quantify and purify exosomes respectively. Due membrane antigen) aptamer, a recognized PSMA target ligand to higher affinity between exosomes and aptamers, ssDNAs with high affinity and low immunogenicity was applied to were replaced and disassociated into supernatant, subsequently capture exosomes 29. And ssDNAs complementary with separated from SMCs by magnet (Figure 1b). In such a pattern, aptamers were divided into two parts in order to decrease the the quantity of released ssDNAs could represent that of hybridization energy with aptamers, and further increase the exosomes. Particularly, ssDNA was comprised of a competition capacity of exosomes to replace ssDNAs from combination and recognition part which is complementary to aptamers, as well as double the exosomes signal (single aptamer and HP1 respectively. HP2 was a molecular beacon exosome produces two ssDNA signal) which could be helpful designed based on Förster resonance energy transfer (FRET) when exosomes concentration was low. According to previous effect between the quencher (BHQ) and fluorescent dye (FITC) studies, 30 two hairpin DNA probes (HP1, HP2) were designed at separate ends of the hairpin structure (Figure 1c). Owing to to initiate amplification cycle and detection of released the stronger affinity, HP2 took place of ssDNAs to form HP1ssDNAs with low concentration. Particularly, HP2 was HP2 complex thereby turning on fluorescence signal. Exactly designed as a molecular beacon, an “on-off” fluorescent probe target ssDNAs allowed the initiation of amplification process, which has been widely used in various biomarkers and produced detection signals. Exosomes separated by detections.31-32 The high sensitivity and efficiency of this magnet were further purified by Endonuclease EcoRI platform were verified in both cell medium and urine samples. corresponding to restriction sites designed in aptamers,
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avoiding the drawbacks of irreversible binding of immunoaffinity capture (Figure 1d). Characterization of superparamagnetic conjunctions. To validate the formulation of SMCs, FTIR absorbance was detected to prove the formation of amido bond between nanoparticles and aptamers. At the same time, the absorbance intensity at 260 nm was used to quantify the aptamers before
Figure 2. Characterization of SMC-MB. (a) FTIR spectrum of Fe3O4 cores and conjunctions. (b)Absorbance intensity in supernatant before and after aptamers reaction with Fe3O4 cores. (c) PAGE gel of different samples: Lane 1: standard DNA markers; Lane 2: 0.5 mM of ssDNA1; Lane 3: 0.5 mM of ssDNA2; Lane 4: supernatant after ssDNA1 incubation; Lane 5: supernatant after ssDNA2 incubation. (d) Absorbance intensity in supernatant before and after ssDNA incubation. (e) Zeta potential of superparamagnetic Fe3O4 cores, Fe3O4-EDC, and Fe3O4 cores-aptamer and SMCs. (f) The hydrodynamic diameters of superparamagnetic Fe3O4 cores and SMCs. (g) TEM image of superparamagnetic Fe3O4 cores. (h) TEM image of superparamagnetic conjunctions (SMCs). and after amide reaction in the supernatant, indirectly hybridization of ssDNA1 and ssDNA2 with PAGE as well as quantification method of aptamers combination. UV-Vis spectrograph. The bands of Lane 2 and Lane 3 Polyacrylamide gel electrophoresis (PAGE) and absorbance referred to two ssDNAs standards before reaction, while Lane detection were aimed to identify ssDNAs hybridization 4 and Lane 5 were supernatant after two ssDNAs reacting with efficiency. As displayed in Figure 2a, the peak shift from 1690 aptamers. The weakened signal suggested successful to 1630 cm-1 were attributed to formation vibration of carbonyl conjugation of ssDNAs and aptamers. The hybridization (C=O) of amido bond. And the peak at 1380 cm-1 and 3100 efficiency calculated by grey value and absorbance peak were cm-1 were ascribed to the bending vibrations of C-N bond and showed in Figure S2, which achieved similar efficiency results stretching vibrations of N-H bond in the conjunction. All the as approximately 70%. The zeta potential of specific groups in the FTIR spectrum indicated the covalent superparamagnetic Fe3O4 cores were about -9.8 mV due to conjunction of aptamers and Fe3O4 cores. The intensity of carboxyl groups functionalized on the surface of Fe3O4 aptamers in supernatant decreased after 12 h reaction, particles. After modified with aptamers and ssDNAs, zeta indicating the binding of aptamers to superparamagnetic Fe3O4 potential decreased gradually to -16 mV and -23 mV (Figure cores, and the binding efficiency was about 80% calculating 2e), which may result from the negative charge of ssDNAs. In other hand, superparamagnetic particle-aptamers and ssDNAs from absorbance peak value (Figure 2b). On the other hand, Fe3O4 cores directly incubated with aptamers without conjunctions exhibited average diameters around 20 nm, as condensing agent caused no decrease in supernatant, which evidenced by dynamic light scattering (DLS) and transmission suggested ignorable non-specifically attachment in the electron microscopy (TEM) in Figure 2f and Figure 2g-h, reaction system (Figure S1).Figure 2c and d described the which indicating that SMCs had ultra-small diameters and the
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binding of aptamers and ssDNAs led to slight increase to Fe3O4 cores, which showed 10 nm in diameter. Superparamagnetic particles with small size were easier redispersed and exhibited better solubility in aqueous solution, as well as stronger magnetism. Instead, some commercial magnetic beads can easily precipitate or form large complexes, which display disadvantages on purification and separation operations. Feasibility of amplification methods for ssDNA detection. The quantity of exosomes in real samples might result in extremely low concentration of replaced ssDNAs. Thus DNA amplification method played a vital role in exosome detection. Taking the advantages of base pairing principle and stem-loop structure of DNA realized the nonenzymatic amplification of target ssDNAs. First of all, hairpin DNA HP1 and HP2, as well as HP1-HP2 complex were determined by PAGE assay, which confirmed the feasibility of the amplification strategy. In the absence of target ssDNAs, no HP1-HP2 complexes were formed, and only target ssDNAs could trigger the amplification circle. Random DNAs were set as contrast group (Figure 3a). Oligo Analyzer 3.1 was used to calculate the relevant parameters Tm (annealing temperature), ΔG (gibbs free energy), ΔH (enthalpy change) and ΔS (entropy change) of hairpin probes HP1 and HP2 (Table S2), and predicted the stability of oligonucleotides. As displayed in Figure S3, the hairpin structures HP1 and HP2 were stable enough at 37 °C with hybridization of 14 and 11 pairs of bases in the stem parts. The Tm value of HP1, HP2 and HP1-HP2 duplex calculated by RNA Structure were 73.3°C, 69.0°C and 71.6°C (CNa+=25mM, CMg2+=4.5mM) , respectively, indicating the stability of HP1 and HP2. Furthermore, the hybridization between bases in single DNA was superior to bases from different DNAs. Additionally, mixture of HP1 and HP2 could regain the fluorescence signal as temperature reached 50°C, revealing the temperature should be carefully controlled (Figure S4). The optimized incubation time and ratio was further discussed in Figure S5 and S6, which was 1 hour for incubation with a ratio of 1.5:1 for HP1 and HP2. To simplify the detection methods of amplified ssDNAs, HP2 was designed as a molecular beacon, which had no fluorescence signal in stem-loop structure owing to the FRET between the fluorescent group FITC and the quenching group BHQ, while regained fluorescence in HP1-HP2 complex because of the remoteness of the two groups. Similarly, the relevant fluorescence at the wavelength of 525 nm indicated the quantity of HP1-H2 complex and was aroused from target DNAs (equimolar mixture of ssDNA1 and ssDNA2). The fluorescence responses of different samples were described in Figure 3b. Obviously, there was a linear relationship between logarithmic target ssDNA concentration and fluorescence intensity (Figure 3c-3d), demonstrating the effectiveness and practicability of the amplification method in ssDNA detection.
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Figure 3. Validation of amplification platform for ssDNA detection. (a) PAGE gel of different samples in the amplification system: Lane 1: standard DNA markers; Lane 2: 1 μM of HP1; Lane 3: 1 μM of HP2; Lane 4: 1 μM of HP1 incubated with 1 μM of HP2 for 1h. Lane 5: 1 μM of HP1 incubated with 1 μM of random DNA and 1 μM of HP2 for 1h. Lane 6: 1 μM of HP1 incubated with 1 μM of target DNA and 1 μM of HP2 for 1h. (b) Fluorescence intensity of molecular beacon in different samples corresponding to (a). (c) The relationship between target DNA concentrations and fluorescence intensity of molecular beacon. (d) The linear relationship between logarithmic target DNA concentrations and fluorescence intensity.
Figure 4. Detection of PCa related exosomes. (a) Dilution and calibration curve of exosomes from LNCaP cells diluted by exosome-free urine and PBS. (b) The linear relationship between fluorescence intensity and logarithmic exosomes count. (c)Fluorescence intensity of exosomes from different cell cultures medium. (d)Western blot of PSMA expression in exosomes from different cell cultures medium. (e) TEM images of SMC-MB captured exosomes.
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PCa related exosomes detection efficiency by SMCMB. Based on the optimized conditions, we developed and characterized an analysis platform for PCa related exosomes with PSMA aptamer. To investigate detection efficiency of the novel platform, LNCaP cell-derived exosomes were enriched by ultracentrifugation and gradient diluted to exact concentrations by exosome-free urine and PBS separately. As shown in Figure 4a, fluorescent signal elevated along with the increasing exosome concentrations, which resulted from increasing ssDNAs released from the SMCs. Similar to the relationship between the concentration of ssDNAs and fluorescence signal of HP1-HP2 complexes (Figure 4b), a linear relationship was obtained between fluorescent signal and the logarithmic exosomes counts ranging from 103 to 108 /μL, and the correlation coefficient (R2) is 0.989. Owing to the interference of urine itself, the limit of detection (LOD) was estimated and calculated to be ~100 particles /μL, much higher than the detection limit in PBS solution(~50 particles /μL) due to the complex urine background. However, in accordance with the results of several studies and our research, the concentration of urine exosomes is far beyond the calculated LOD in this experiment, revealing the applicability in urine exosomes detection. Furthermore, it should be noted that the dual function platform described here is simple and highefficient without complicated instruments and expensive reagents. To further explorethe specificity of the platform, five cancer cell derived ex-osomes were incubated with SMC, and detected by MB ampli-
Figure 5. Detection of PCa related exosomes. (a) Dilution and calibration curve of exosomes from LNCaP cells diluted by exosome-free urine and PBS. (b) The linear relationship between fluorescence intensity and logarithmic exosomes count. (c)Fluorescence intensity of exosomes from different cell cultures medium. (d)Western blot of PSMA expression in exosomes from different cell cultures medium. (e) TEM images of SMC-MB captured exosomes. fication cycle. Fluorescence intensity of exosomes from Exosome isolation efficiency compared to human prostate cancer cell LNCaP was significantly higher ultracentrifugation. With the benefit of restriction site of than other cancer cell A549, T24, HCT116 and HepG2 (Figure aptamers, exosomes captured by SMCs could be easily 4c), consistent with the protein expression levels of PSMA separated from particles. And the morphology and size (Figure 4d), which also proved aptamers applicability in this distribution of exosomes isolated by SMCs followed by platform. Figure 4e displayed typical binding morphology of enzyme restriction were determined by TEM and DLS. SMCs and exosomes, indicating successful capture of Considering ultracentrifugation (UC) is the gold standard exosomes by SMCs. isolation method in current exosome studies, which is employed by over 50% researchers, ultracentrifugation group
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was utilized to compare with SMC-MB platform to further illustrate capture capacity. Thus, LNCaP cell derived exosomes were isolated by UC, and further resuspended in exosomes-free urine followed by SMC-MB treatment. Generally, UC and SMCs isolated exosomes were both capshaped with average diameters of 30-150 nm (Figure 5a, 5b). However, exosomes in UC precipitate inevitably existed with protein aggregations and extracellular vesicles (EVs, more than 200 nm in diameter), leading to complex background and larger diameter determined by DLS (Figure 5c). On the contrary, exosomes obtained by SMCs isolation method were observed fewer impurities and smaller size. Also, the total protein and particle concentration of exosomes was analyzed by BCA protein determination and NTA in both UC and SMC-MB group. As a result, more protein was acquired by UC (Figure 5d). It may be induced by protein bundles and EVs resulting from non-specific isolation method. Meanwhile, there was no big difference of particle concentration between two groups. Exosome-specific proteins PSMA, CD63, and CD9 detected by western blot further confirmed the successful exosomes isolation, (Figure 5e) displaying the exosomes isolation capability of SMC-MB platform PCa detection efficiency via SMC-MB platform. Considering the platform exhibits excellent properties of exosome isolation and detection, SMC-MB was further applied to capture and detect exosomes related to prostate cancer (PCa) in urine samples. Prostate specific membrane antigen (PSMA) was regarded as a specific prostate cancer (PCa) biomarker in a number of researches, with higher expression in prostate cancer. First of all, immunogold labeling of PSMA in exosomes from urine of healthy male and prostate cancer subjects were demonstrated by TEM in Figure 6a and 6b. Apparently, PSMA antibody labeled goldnanoparticles anchored on some of exosomes from PCa patients while irregularly distributed in cancer-free samples. Accordingly, SMC-MB platform mentioned above were directly applied to analyze PSMA-positive exosomes in urine samples of PCa patients and healthy donors, and evaluated the diagnosis efficiency of PCa. Exosomes separated by SMC platform also matched the established exosomal morphology and size. The PSMA, CD63 and CD9 protein expressions were determined to explain the validity of SMC-MB platform. It was obvious that PSMA-positive exosomes also harbored exosomes specific proteins. However, since our platform was designed for PSMA positive exosomes, which was extremely low in healthy donors, specific protein expressions of exosomes captured by SMC in healthy urines were hard to be detected as showed in Figure.6c.To further explore the efficiency of PSMA-positive exosomes in urine for PCa diagnosis, we collected urine samples from twenty PCa patients and fifty healthy donors. The fluorescent signal of PCa group obtained from SMC-MB detection platform was significantly higher than healthy group (P
