Materials
Anhydrous ferric chloride, NaAc, magnesium chloride hexahydrate (MgCl2.6H2O), (3-aminopropyl)-triethoxysilane (APTES), tetraethylorthosilicate (TEOS, 98%), poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA), and ammonium hydroxide (NH3·H2O, 28%) were obtained from Aladdin Reagents (China). Ethylene glycol (EG) was pretreated with a sufficient molecular sieve (type 3). Succinic anhydride (SA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). 2-Morpholinoethanesulfonic acid (MES) was obtained from Solon (USA). Anti-CD63 antibody (ab59479) and fluorescein isothiocyanate (ab18235)-labeled anti-CD63 antibody were purchased from Abcam (USA). BSA was purchased from Roche (USA). The human CEA ELISA Kit and human GPC-3 ELISA Kit were purchased from Jianglai Biotechnology (China). The water employed in all experiments was deionized and ultrafiltered by a Millipore Milli-Q system with minimum resistivity of 18 MΩ · cm.
Synthesis of Fe3O4 nanoparticles
Fe3O4 nanoparticles were synthesized via a “one-pot” hydrothermal method. First, 1.6 mmol of FeCl3 (0.26 g), 8.8 mmol of NaAc (1.2 g), 1.8 mmol of PSSMA (0.4 g), and a moderate amount of water (20–100 μL) were added to 16 mL EG pretreated by a molecular sieve. The mixture was stirred for ~30 min, and the process continued after 6 mmol of solid NaOH (0.24 g) was added. The resultant mixture was transferred to a 20 mL autoclave to heat in a muffle furnace for 9 h at 190 °C.
Synthesis of Fe3O4@SiO2 nanoparticles
The synthesized magnetic Fe3O4@PSSMA particles were collected with a magnet, washed three times with ethanol, and dispersed in 12 mL water. Then, 40 mL ethanol and 2 mL ammonium hydroxide were added to 6 mL of the Fe3O4@PSSMA and mixed well. The mixture was sonicated for ~10 min and then transferred to a 100 mL three-neck flask while stirring at 6000 rpm in a water bath at 50 °C. After the mixture was stirred vigorously for 10 min, 200 μL of TEOS was added dropwise to the brown mixture. The prepared Fe3O4@SiO2 nanoparticles were washed with ethanol and water after stirring for an hour and stored with 2 mL of ethanol for further use.
Preparation of Fe3O4@MgSiO3
First, 0.25 mmol of MgCl2.6H2O and 3.3 mmol of NH4Cl were added to 10 mL of an aqueous solution, and ammonia solution (500 μL, 28%) was added to the Fe3O4@SiO2 dispersion prepared above. The mixture was then transferred to an autoclave, heated to 140 °C, and maintained for ~14 h. The brown precipitate was collected with a magnet, washed three times, and dispersed in moderate ethanol for further applications.
Characterization
The geometry and size of the Fe3O4@MgSiO3 nanoparticles were observed by scanning electron microscopy (SEM, Hitachi, SU8010, Japan). The nanostructures of the Fe3O4@MgSiO3 nanoparticles were observed through transmission electron microscopy (TEM, FEI, Talos, USA). The distribution of particle size and surface charges was measured on a Zetasizer Nano ZS (Malvern, ZEN3600, U.K). The phase composition of the Fe3O4@MgSiO3 nanoparticles was measured by X-ray diffraction (XRD, Bruker, D8 Advance, Germany). To compare them to those of the Fe3O4@SiO2 nanoparticles, the BET surface area and pore size distribution of the Fe3O4@MgSiO3 nanoparticles were evaluated through N2 adsorption-desorption measurements at 77 K (Micromeritics 3Flex, USA). The magnetic properties were determined by a vibrating sample magnetometer (VSM, PPMS DynaCool, USA).
Antibody modification
The CD63 antibody was immobilized on the surface of the Fe3O4@MgSiO3 nanoparticles through covalent modification. The NaOH, 2% APTES-ethanol solution, and 10% succinic anhydride (SA) solution treatment of Fe3O4@MgSiO3 nanoparticles modified them with amino and carboxylate groups. Then, the combination of the Fe3O4@MgSiO3 nanoparticles and CD63 antibody was completed after 3 h of incubation at 37 °C in EDC/NHS solution. Finally, the CD63 antibody-modified nanoparticles were obtained after being washed three times with buffer solution and blocked with 1% BSA. The FITC-labeled anti-CD63 antibody (ab18235) served to verify the modification with a concentration range of 0.1–50 μg/mL. After incubation with the Fe3O4@MgSiO3 nanoparticles and washing three times, they were observed with optical and fluorescence microscopy. To avoid interfering with the detection of fluorescence of the exosomes, we subsequently used the CD63 antibody (ab59479) to modify the nanoparticles in a concentration range from 0.001 to 10 μg mL−1.
Preparation of exosome standards
HepG2 cells were cultivated in DMEM containing 10% FBS and 1% penicillin–streptomycin in an incubator maintained at 5% CO2 and 37 °C and then cultivated in DMEM containing exosomal-free FBS for 48 h. Then, the supernatant of the culture was centrifuged at 300×g and 2000×g for 10 min. After filtration through a 0.22 μm filter, the supernatant of the culture was separated by centrifugation at 120,000×g (70 min × 2) at 4 °C. The precipitated exosome pellets were resuspended in 200 μL PBS buffer and stored at −80 °C to form exosome standards. The average nanoparticle tracking analysis (NTA) value of the prepared exosome standards was measured to be 7.4 ± 0.38 × 109 particles mL−1, and their morphology was observed by TEM.
Enrichment and isolation of exosomes using Fe3O4@MgSiO3 nanoparticles
After using a 1.5 μg CD63 antibody to modify 3 mg Fe3O4@MgSiO3 nanoparticles, the modified nanoparticles were coincubated with 50 μL exosomes at 37 °C on a shaker. During this process, the CD63 antibody modified on the nanoparticles became bound to the specific proteins on the exosomes. Then, they could be isolated from the solution after they were combined by applying a magnetic field for 10 s. The Fe3O4@MgSiO3 nanoparticles with captured exosomes were washed with PBS three times, and the membrane dye PKH26 with a concentration of PKH26 of 2 × 10−6 was used to stain the exosomes to indicate their capture. The fluorescence intensity was detected with a microplate reader at an excitation wavelength of 551 nm.
To optimize the exosome capture conditions, the number of exosomes before and after capture was measured with BCA to quantitatively calculate the capture efficiency. The Fe3O4@MgSiO3 nanoparticles were modified with CD63 antibodies at different concentrations (0.1, 1, 10, 100, and 1000 µg mL−1), and then the Fe3O4@MgSiO3 nanoparticles were incubated with identical concentrations of exosomes under the same capture time (30 min).
Western blot analysis of exosomes
Western blotting was applied to verify the specific marker protein CD63 in exosomes captured on nanoparticles and the standards, which were pretreated with RIPA lysis buffer on ice for 15 min. The loading buffer was mixed and heated for 5 min at 100 °C. Then, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out. After transmembrane incubation, the membrane was incubated overnight at 4 °C with a primary antibody (1:1000) against CD63 and then incubated with a secondary antibody for one hour. The image was obtained by a gel image system.
Detection for captured exosomes
An HRP-assisted immunosorbent assay was employed to detect the protein biomarkers of exosomes. In this process, 100 µL HRP-CEA or HRP-GPC was incubated with nanoparticles that captured the exosomes containing protein markers together at 37 °C for 1 h, forming antibody-antigen-antibody complexes. Then, excess enzyme-labeled antibodies were washed away, and 50 µL substrate TMB was added to develop color by incubating for 15 min at 37 °C in the dark. After 50 μL stop solution was quickly dripped in the well, the content of CEA or GPC could be obtained by measuring the absorbance at 450 nm.
Clinical samples
Two milliliters of clinical samples from patients with liver cancer (n = 10) and healthy donors (n = 10) were collected at the First Affiliated Hospital of Wenzhou Medical University with informed consent (Wenzhou, China). These blood samples were pretreated with centrifugation at 4 °C for 15 min (4000 × g). After they were captured, the exosomes isolated from clinical samples were detected using the Fe3O4@MgSiO3 nanoparticles.
Statistical analysis
All data are shown as the means ± standard deviation. Statistical significance was assessed by a two-tailed Student’s t-test. Values of p < 0.05 were judged statistically significant.
