Tatu Rojalin

Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector

AbstractIn this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences. Graphical abstractTime-resolved Raman measurement of a pharmaceutical sample using the complementary metal-oxide semiconductor (CMOS) single photon avalanche diode (SPAD) detector technology

FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel

ABSTRACT In response to physiological and artificial stimuli, cells generate nano‐scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane‐derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti‐cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel‐containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real‐time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next‐generation targeted drug delivery systems. Graphical abstract Figure. No caption available. HighlightsA single probe reveals more when instead fluorescence intensity its lifetime is usedThree different steps of drug delivery process can be recognized for the FLIM dataUp‐take pathways of extracellular vesicle subpopulations are distinguished with FLIMExosomes deliver the drug mainly by endocytosisMicrovesicles deliver the drug by both endocytosis and cell membrane fusion

Oligomerization Alters Binding Affinity between Amyloid Beta and a Modulator of Peptide Aggregation

The soluble oligomeric form of the amyloid beta (Aβ) peptide is the major causative agent in the molecular pathogenesis of Alzheimers disease (AD). We have previously developed a pyrroline-nitroxyl fluorene compound (SLF) that blocks the toxicity of Aβ. Here we introduce the multi-parametric surface plasmon resonance (MP-SPR) approach to quantify SLF binding and effect on the self-association of the peptide via a label-free, real-time approach. Kinetic analysis of SLF binding to Aβ and measurements of layer thickness alterations inform on the mechanism underlying the ability of SLF to inhibit Aβ toxicity and its progression towards larger oligomeric assemblies. Depending on the oligomeric state of Aβ, distinct binding affinities for SLF are revealed. The Aβ monomer and dimer uniquely possess sub-nanomolar affinity for SLF via a non-specific mode of binding. SLF binding is weaker in oligomeric Aβ, which displays an affinity for SLF on the order of 100 μM. To complement these experiments we carried out molecular docking and molecular dynamics simulations to explore how SLF interacts with the Aβ peptide. The MP-SPR results together with in silico modeling provide affinity data for the SLF-Aβ interaction and allow us to develop a new general method for examining protein aggregation.

Multimodal Nonlinear Optical Imaging for Sensitive Detection of Multiple Pharmaceutical Solid-State Forms and Surface Transformations

Two nonlinear imaging modalities, coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG), were successfully combined for sensitive multimodal imaging of multiple solid-state forms and their changes on drug tablet surfaces. Two imaging approaches were used and compared: (i) hyperspectral CARS combined with principal component analysis (PCA) and SFG imaging and (ii) simultaneous narrowband CARS and SFG imaging. Three different solid-state forms of indomethacin-the crystalline gamma and alpha forms, as well as the amorphous form-were clearly distinguished using both approaches. Simultaneous narrowband CARS and SFG imaging was faster, but hyperspectral CARS and SFG imaging has the potential to be applied to a wider variety of more complex samples. These methodologies were further used to follow crystallization of indomethacin on tablet surfaces under two storage conditions: 30 °C/23% RH and 30 °C/75% RH. Imaging with (sub)micron resolution showed that the approach allowed detection of very early stage surface crystallization. The surfaces progressively crystallized to predominantly (but not exclusively) the gamma form at lower humidity and the alpha form at higher humidity. Overall, this study suggests that multimodal nonlinear imaging is a highly sensitive, solid-state (and chemically) specific, rapid, and versatile imaging technique for understanding and hence controlling (surface) solid-state forms and their complex changes in pharmaceuticals.

Time-resolved SERS for characterizing extracellular vesicles

The aim of this work is to develop a platform for characterizing extracellular vesicles (EV) by using gold-polymer nanopillar SERS arrays simultaneously circumventing the photoluminescence-related disadvantages of Raman with a time-resolved approach. EVs are rich of biochemical information reporting of, for example, diseased state of the biological system. Currently, straightforward, label-free and fast EV characterization methods with low sample consumption are warranted. In this study, SERS spectra of red blood cell and platelet derived EVs were successfully measured and their biochemical contents analyzed using multivariate data analysis techniques. The developed platform could be conveniently used for EV analytics in general.

Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content(Conference Presentation)

Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four “subpopulations” of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.