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Nano and Micro Technologies

Nano and Micro Biosensing Technologies for Disease Diagnosis and Monitoring

In Situ Profiling of RNA and Proteins in Single Extracellular Vesicles and Particles for Intravesicular, Intervesicular, and Interparticle Heterogeneity Analyses

Saturday, October 14, 2023
2:45 PM – 3:00 PM PDT
Location: 337 (Center, Level 3)
Introduction:: Introduction: Extracellular vesicles (EVs) are membranous particles released by cells that are implicated in intercellular communication and contain bioactive proteins, lipids, polysaccharides, and nucleic acids. Due to their presence in all biofluids, EVs present a promising source for minimally invasive biomolecular diagnostics for various diseases. However, EVs are molecularly heterogeneous and are rare compared to the abundance of lipoproteins (LPs), which are cholesterol-transporting particles present in complex biofluids, particularly in blood. Given their physical similarity, isolation methods co-isolate the particles and are irreproducible insofar as yielding technique-dependent profiles. On the other hand, the molecular analysis of EVs and particles (EVPs) in bulk requires lysis to access the intraluminal contents, diluting the biomarkers and reducing detection sensitivities. Various optical and non-optical techniques have been adapted to characterize single EVs (siEVs). However, the methods are laborious, low-throughput, or incompatible with molecular profiling. In situ detection of siEVs has augmented sensitivities and aided in qualitatively demonstrating intravesicular heterogeneity via multiplexed protein detection. While in situ detection of multiple protein targets on siEVs is readily performed, accessing the intraluminal cargo and detecting multiple targets without altering the native structure of single EVPs (siEVPs) has not been achieved. Herein, we demonstrate a novel method for the single-particle detection of RNA and protein in EVs and LPs on controllable microdomains to highlight intravesicular, intervesicular, and interparticle heterogeneity.


Materials and Methods:: Materials and Methods: A microarray of domains for siEVP capture was generated by photoetching a non-biofouling polymer monolayer on a glass coverslip with ultraviolet (UV) light in distinct surface positions, allowing for the adsorption of proteins onto the exposed regions. Avidin was adsorbed to the distinct surface positions promoting their subsequent binding to free biotinylated antibodies against epitopes on EVs and LPs to positively enrich siEVPs (Fig. 1A). The level of photoetching was mediated by a digital micromirror device (DMD)-based UV projection system for the controllable degradation of the non-biofouling monolayer. Intravesicular heterogeneity investigations were performed by marrying two molecular detection methods, immunofluorescence and fluorescence in-situ hybridization, to simultaneously detect proteins and RNAs in non-lysed siEVPs. The signals were detected utilizing a total internal reflection fluorescence microscopy, which generates an exponentially decaying wavefront for the detection of fluorophores near the coverslip surface. Exogenous miRNAs were transfected into cells via electroporation whereby the exogenous miRNAs were naturally packaged in EVs for robust analyses on RNA specificity (Fig. 1B). On the other hand, intervesicular and interparticle investigations were performed by customizing the antibodies utilized to immobilize the siEVPs and probe their molecular content. Furthermore, LPs were isolated from human serum via dextran-based precipitation for single LP (siLP) investigations.


Results, Conclusions, and Discussions:: Results: Being at the forefront of in situ RNA detection within the intraluminal space of siEVPs, an in-depth characterization of exogenous miRNA transfected in human EVs was carried out, whereby three miRNA species were co-detected in single non-lysed EVs at a rate of ~31% for two targets and 5.5% for three targets. Further intravesicular heterogeneity analyses were performed by colocalizing different regions of an mRNA strand, multiple tetraspanins, and protein-RNA interspecies detection with rates ranging from 19-29% for two targets and 2-3% for three targets (Fig. 1C). For intervesicular heterogeneity, EVs were immobilized by targeting ARF6, annexin A1, CD63, CD9, and EGFR whereby nine biomarkers spanning protein, mRNA, and miRNA were analyzed. Multivariate analyses illustrated the vast heterogeneity of EVs. By clustering the biomolecules into species, similarities were observed between CD63+/CD9+ and ARF6+/annexin A1+ subpopulations (Fig. 1D). We further subjected EVs and serum-isolated LPs on microdomains functionalized to capture ApoB+/ApoA1+ and CD63+/CD9+ siEVPs. While LPs and EVs co-isolated during the purification of EVPs, interestingly, we found the co-isolation of LPs and EVs at the single-particle level, where single non-lysed LPs were immobilized on anti-CD63/CD9 decorated microdomains and expressed ApoB/CD63 (Fig. 1E).


Conclusions and Discussions: Progressing towards single-particle profiling of siEVPs without compromising the native structure of the particles, ensured enhanced sensitivities in profiling biomolecular expression, outperforming conventional bulk-analysis methods, such as ELISA and qRT-PCR. Furthermore, the use of in situ detection afforded unique perspectives into intravesicular heterogeneity via colocalization analyses and intervesicular and interparticle heterogeneity via multivariate experimental design, which is obscured in bulk-analysis methods. With the enhanced sensitivity and the tunability of the assay, we discovered that blood-derived particles not only co-isolate at the bulk level but also co-isolate as single-particle complexes, which concurs with newer evidence on the complexity of siEVPs. Many investigations underscore this complexity and assume that LPs are sorted out by positive immunoaffinity, indicating the necessity for siEVP methods. The success of this work opens avenues in investigating EVP heterogeneity for biomarker discovery, diagnostics, biogenesis investigations, and deconvolution of EVP heterogeneity.


Acknowledgements (Optional): : This work was supported by the U.S. National Institutes of Health (NIH) grants UG3/UH3TR002884 (ER) and U18TR003807 (ER, LJL). Additional funding was provided by the William G. Lowrie Department of Chemical and Biomolecular Engineering and the Comprehensive Cancer Center at The Ohio State University (ER).

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