Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50–150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70–500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10–70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with 129Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.


NTA and immuno-electron microscopy
Initially, the concentrations and size distributions of the EV samples were characterized by NTA using the Malvern Panalytical NanoSight NM300 instrument equipped with a 405 nm laser. According to NTA, the concentrations varied between 10 10 and 10 12 particles/ml (see Table S1 in ESI †). Some EV samples were also analyzed by transmission electron microscopy (TEM). 2 µl of each sample were deposited on a Formvar carbonated grid (glow-discharged) and after negative staining with 2% uranyl acetate and immunostaining with anti-CD63 antibody (LAMP-3, MBL, Nagoya, Japan) examined using the Tecnai G2 Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands). Protein A-gold complex (10 nm) served to detect the primary anti-CD63 antibody. Images were captured with a charge-coupled device camera (Quemesa, Olympus Soft Imaging Solutions GMBH, Münster, Germany).

RNA isolation from EVs
RNA from milk samples was extracted using a modified Total exosome RNA isolation (Invitrogen) protocol.
Samples were suspended in PBS overnight on slow rocking at + 4˚C. Samples were mixed with the lysis buffer and incubated on ice for 5 minutes. Equal volume of acid-phenol-chloroform mixture was added and mixed by slowly inverting for 3 minutes. After incubation samples were centrifuged for 5 minutes with maximum speed with Eppendorf centrifuge 5415 D. Upper phase was extracted and mixed with 1.25 times the volume of 100 % ethanol. Samples were centrifuged through purification filter cartridges and washed three times in total with wash solution 1 and 2/3. Samples were eluted with 2 times 50 µl of RNAse free water. Quality was analyzed with Bioanalyzer 2000 RNA 6000 Pico kit (Agilent) and quantity was analyzed with Qubit 4 and microRNA assay (Thermo Scientific).
For RNA isolation from cell-derived EVs 600 µl of Qiazol (Qiagen) was added to 100 µl of EVs and mixed by vortexing. After 2-5 minutes incubation at RT 90 µl chloroform was added, then extraction of RNA was done according to ExRNeasy (Qiagen) protocol using minElute RNA column. RNA was eluted in 14 µl water, 1 ul was used for profiling with bioanalyzer pico 6000 chips using total eukaryotic RNA program.
Supplementary results and discussion DOSY and NTA particle size distributions is the chemical shift of the CEST response, Γ the full width at half maximum, and the depolarization rate for on-resonant saturation (∆ = ). R represents a Rician noise floor that originates from integrating the Xe spectra of dissolved Xe and yields values ≠ 0. Γ is determined by the applied saturation power 1 and the exchange rate BA, from each CEST pool (Bi) into the free solution pool (A): Γ = 2 √ ( 1 ) 2 + ( BA, ) 2 . This assumes that no significant transverse relaxation rate contributes to the width of the CEST responses. This condition is usually at least known for the CEST signal of CrA-ma in water. Then, the depolarization rate ≈ B, BA ( 1 ) 2 ( BA, ) 2 +( 1 ) 2 can be used to obtain the fraction of bound Xe in each pool, B, , (relative to the pool of free Xe) after the exchange rate has been determined from the width Γ . The fit does not require to normalize the data and leaves the starting magnetization S0 as free parameter. The CEST response from excess CrA in solution is represented by ℒ 1 , while ℒ 2 and ℒ 3 describe the responses from CrA in larger lipid vesicles (previously described signal with ca. 10 ppm downfield shift) and the newly discovered signal from CrA in the lipoprotein/exomer environment.
The applied saturation power of 2.5 mW corresponds to 1 = 5.69 µT and should yield a minimum line width of Γ ≈ 1.22 ppm. A completely free fit yields an underestimated Γ 3 = 0.94 ppm (blue fitting line). This value was thus restricted to at least 1.22 ppm and the fitting was repeated (red spectrum). Fitting results are shown in Table S2.  Figure 6). M -protein ladder (marker).

Fig. S8
Western blot of milk EV samples, isolated by sequential ultracentrifugation (UCF) or by using filtration (see Materials and methods), with antibodies against common EV markers ALIX, TSG101, and CD81. Milk samples before EV isolation were used as controls.