RESUMO
Molecular exchange between different physical or chemical environments occurs due to either diffusion or chemical transformation. Nuclear magnetic resonance (NMR) spectroscopy provides a means of understanding the molecular exchange in a noninvasive way and without tracers. Here, we introduce a novel two dimensional, single-scan ultrafast Laplace NMR (UF LNMR) method to monitor molecular exchange using transverse relaxation as a contrast. The UF T2-T2 relaxation exchange spectroscopy (REXSY) method shortens the experiment time by one to two orders of magnitude compared to its conventional counterpart. Contrary to the conventional EXSY, the exchanging sites are distinguished based on T2 relaxation times instead of chemical shifts, making the method especially useful for systems including physical exchange of molecules. Therefore, the UF REXSY method offers an efficient means for quantification of exchange processes in various fields such as cellular metabolism and ion transport in electrolytes. As a proof of principle, we studied a halogen-free orthoborate based ionic liquid system and followed molecular exchange between molecular aggregates and free molecules. The results are in good agreement with the conventional exchange studies. Due to the single-scan nature, the method potentially significantly facilitates the use of modern hyperpolarization techniques to boost the sensitivity by several orders of magnitude.
Assuntos
Líquidos Iônicos , Difusão , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodosRESUMO
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.
RESUMO
Ultrafast Laplace NMR (UF-LNMR) reduces the experiment time of multidimensional relaxation and diffusion measurements to a fraction. Here, we demonstrate a method for nonlinear (in this case logarithmic) sampling of the indirect dimension in UF-LNMR measurements. The method is based on the use of frequency-swept pulses with the frequency nonlinearly increasing with time. This leads to an optimized detection of exponential experimental data and significantly improved resolution of LNMR parameters.