(470d) Ultraviolet Photopatterning to Profile of Single Extracellular Vesicles and Particles

Introduction: Extracellular vesicles (EVs), which are membranous particles containing bioactive proteins, lipids, polysaccharides, and nucleic acid, are released by cells and are implicated in interorgan communication. EVs are molecularly and biogenetically heterogeneous and rare compared to other extracellular particles (EPs), such as lipoproteins (LPs), which are cholesterol-transporting particles. Isolation methods tend to co-isolate particles of similar areas and densities, yielding technique-dependent biomolecular profiles. Traditional bulk-analysis methods require lysis to access intraluminal contents and dilute the biomarkers into the transcriptome and proteome of all particles in the sample, leading to averaged biomolecular readouts. Therefore, optical and non-optical single-EV techniques have been developed to better represent the heterogeneity of EVs and must both be performed to verify the presence of single EVs. The in-situ detection of single EVs enhanced sensitivities and qualitatively demonstrated intravesicular heterogeneity via multiplexed protein detection. Accessing the intraluminal space without altering the native structure of single EVs is necessary to colocalize protein with RNA signals. Although micropatterning facilitates the pinpointing of EV capture and provides rapid quality control of non-specificity, microstamping and bioprinting are limited in resolution and pattern design. Therefore, developing a scalable micropatterning method capable of tunable design, multiplexing, and orthogonal measurements is necessary.

Materials and Methods: Micropatterns were generated by photoetching a non-biofouling polymer monolayer on a glass coverslip with ultraviolet (UV) light in distinct surface positions. The photoetched regions allow for the adsorption of proteins and EPs onto the exposed regions. For subpopulation-based sorting, avidin was adsorbed to the distinct surface positions followed by biotinylated antibodies against epitopes on EVs and LPs. The degree of photoetching was mediated via a digital micromirror device (DMD)-based UV illumination that corresponds to photon flux. Intravesicular heterogeneity investigations were performed by marrying immunofluorescence and in-situ fluorescence hybridization in the presence of a Tris EDTA buffer that fluidizes the lipid-bilayer membrane and maintains EV integrity. Exogenous miRNAs were transfected into cells via and naturally packaged in EVs to ensure RNA specificity. Lastly, asymmetric micropatterns were developed to map transmission electron microscopic (TEM) images to total internal reflection fluorescence microscopic (TIRFM) images to provide orthogonal measurements of optical and non-optical measurements of a single EV.

Results: Given the inability of micropatterning and bioprinting to produce non-binary patterns, photon flux was tuned via DMD-based UV illumination, which resulted in gradient micropatterns of single EVs. Various micropattern designs spanning from linear, logarithmic, and Gaussian gradient micropatterns were tested with large EVs (lEVs), small EVs (sEVs), outer membrane vesicles (OMVs), genetically modified EVs, and exomeres, all of which followed the mathematical functions. To better comprehend the kinetics of adsorption, sEVs, lEVs, and cationic lipid nanoparticles were deposited on the surface, revealing a charge-based capture with faster kinetics for sEVs, which was confirmed by computational simulations. To colocalize signals and investigate intravesicular heterogeneity, 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. The success of the technique enabled the first reported colocalization of miRNA, mRNA, and protein in sEVs. To measure intervesicular heterogeneity, EVs were immobilized by targeting ARF6, annexin A1, CD63, CD9, and EGFR and detected for nine biomarkers. Multivariate analyses confirmed the vast heterogeneity of EVs and revealed some similarities between CD63⁺/CD9⁺ EV and ARF6⁺/annexin A1⁺ EV subpopulations. Given the frequent co-isolation of LPs and EVs during the purification, we tested whether CD63/CD9-mediated capture excluded LPs. Surprisingly, LPs and EVs co-isolate at the single-particle level, which we confirmed as EVs with apolipoprotein corona. Due to our selection of probe targets, the possibility exists for EVs captured on the micropatterned surface that produce false-negative signals. Through the one-to-one mapping of TIRFM and TEM images, we were able to superimpose morphological characteristics to fluorescence data, whereby we discovered a majority of signals from genetically enhanced fluorescent EVs lacked fluorescence, highlighting the necessity to perform orthogonal measurements at a single-EV resolution.

Conclusions and Discussions: Actuating the adsorption properties of the coverslip surface through UV illumination enabled the precise control of EV micropatterning. The control enabled by DMD-based projections warranted gradient designs, facile colocalization of biomolecular species, and the superimposition of optical and non-optical modalities at the single-EV resolution. The success of this work paves the way to deconvolute the heterogeneity of EVs, which can be designed to reveal the packaging mechanisms of cargo, illuminate EV biogenesis pathways, and produce highly sensitive diagnostics.