Methods to analyze extracellular vesicles at single particle level

Micro and Nano Systems Letters - Tập 10 - Trang 1-13 - 2022
Yongmin Kwon1, Jaesung Park1,2
1Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea

Tóm tắt

Extracellular vesicles (EVs) are nano-sized vesicles derived from cells that transport biomaterials between cells through biofluids. Due to their biological role and components, they are considered as potential drug carriers and for diagnostic applications. Today's advanced nanotechnology enables single-particle-level analysis that was difficult in the past due to its small size below the diffraction limit. Single EV analysis reveals the heterogeneity of EVs, which could not be discovered by various ensemble analysis methods. Understanding the characteristics of single EVs enables more advanced pathological and biological researches. This review focuses on the advanced techniques employed for EV analysis at the single particle level and describes the principles of each technique.

Tài liệu tham khảo

Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383. https://doi.org/10.1083/jcb.201211138 van Dommelen SM, Vader P, Lakhal S, Kooijmans SA, van Solinge WW, Wood MJ, Schiffelers RM (2012) Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J Control Release 161(2):635–644. https://doi.org/10.1016/j.jconrel.2011.11.021 Li XH, Zhang J, Li DF, Wu W, Xie ZW, Liu Q (2020) Physiological and pathological insights into exosomes in the brain. Zool Res 41(4):365–372. https://doi.org/10.24272/j.issn.2095-8137.2020.043 van Blitterswijk WJ, Emmelot P, Hilkmann HAM, Hilgers J, Feltkamp CA (1979) Rigid plasma-membrane-derived vesicles, enriched in tumour-associated surface antigens (MLr), occurring in the ascites fluid of a murine leukaemia (GRSL). Int J Cancer 23(1):62–70. https://doi.org/10.1002/ijc.2910230112 Friend C, Marovitz W, Henie G, Henie W, Tsuei D, Hirschhorn K, Holland JG, Cuttner J (1978) Observations on cell lines derived from a patient with Hodgkin’s disease. Cancer Res 38(8):2581–2591 Piffoux M, Silva AKA, Wilhelm C, Gazeau F, Tareste D (2018) Modification of extracellular vesicles by fusion with liposomes for the design of personalized biogenic drug delivery systems. ACS Nano 12(7):6830–6842. https://doi.org/10.1021/acsnano.8b02053 Vogel R, Savage J, Muzard J, Camera GD, Vella G, Law A, Marchioni M, Mehn D, Geiss O, Peacock B, Aubert D, Calzolai L, Caputo F, Prina-Mello A (2021) Measuring particle concentration of multimodal synthetic reference materials and extracellular vesicles with orthogonal techniques: who is up to the challenge? J Extracell Vesicles 10(3):e12052. https://doi.org/10.1002/jev2.12052 Kang H, Kim J, Park J (2017) Methods to isolate extracellular vesicles for diagnosis. Micro Nano Syst Lett. https://doi.org/10.1186/s40486-017-0049-7 Doyle LM, Wang MZ (2019) Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. https://doi.org/10.3390/cells8070727 Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, Arab T, Archer F, Atkin-Smith GK, Ayre DC, Bach JM, Bachurski D, Baharvand H, Balaj L, Baldacchino S, Bauer NN, Baxter AA, Bebawy M, Zuba-Surma EK (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750. https://doi.org/10.1080/20013078.2018.1535750 Min L, Wang B, Bao H, Li X, Zhao L, Meng J, Wang S (2021) Advanced nanotechnologies for extracellular vesicle-based liquid biopsy. Adv Sci (Weinh) 8(20):e2102789. https://doi.org/10.1002/advs.202102789 Chiang CY, Chen C (2019) Toward characterizing extracellular vesicles at a single-particle level. J Biomed Sci 26(1):9. https://doi.org/10.1186/s12929-019-0502-4 Bagci C, Sever-Bahcekapili M, Belder N, Bennett APS, Erdener SE, Dalkara T (2022) Overview of extracellular vesicle characterization techniques and introduction to combined reflectance and fluorescence confocal microscopy to distinguish extracellular vesicle subpopulations. Neurophotonics 9(2):021903. https://doi.org/10.1117/1.NPh.9.2.021903 Lipson A, Lipson SG, Lipson H (2010) Optical physics. Cambridge University Press, Cambridge. https://doi.org/10.1017/CBO9780511763120 Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL (2011) Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7(6):780–788. https://doi.org/10.1016/j.nano.2011.04.003 Sage D, Kirshner H, Pengo T, Stuurman N, Min J, Manley S, Unser M (2015) Quantitative evaluation of software packages for single-molecule localization microscopy. Nat Methods 12(8):717–724. https://doi.org/10.1038/nmeth.3442 Jimenez A, Friedl K, Leterrier C (2020) About samples, giving examples: optimized single molecule localization microscopy. Methods 174:100–114. https://doi.org/10.1016/j.ymeth.2019.05.008 Shen H, Tauzin LJ, Baiyasi R, Wang W, Moringo N, Shuang B, Landes CF (2017) Single particle tracking: from theory to biophysical applications. Chem Rev 117(11):7331–7376. https://doi.org/10.1021/acs.chemrev.6b00815 Deschout H, Cella Zanacchi F, Mlodzianoski M, Diaspro A, Bewersdorf J, Hess ST, Braeckmans K (2014) Precisely and accurately localizing single emitters in fluorescence microscopy. Nat Method 11(3):253–266. https://doi.org/10.1038/nmeth.2843 Stehr F (2021) Advancing quantitative DNA-mediated single-molecule fluorescence microscopy (Doctoral dissertation, lmu). https://doi.org/10.5282/edoc.29117 Rose KA, Molaei M, Boyle MJ, Lee D, Crocker JC, Composto RJ (2020) Particle tracking of nanoparticles in soft matter. J Appl Phys. https://doi.org/10.1063/5.0003322 Bian X, Kim C, Karniadakis GE (2016) 111 years of Brownian motion. Soft Matter 12(30):6331–6346. https://doi.org/10.1039/c6sm01153e Kim A, Ng WB, Bernt W, Cho NJ (2019) Validation of size estimation of nanoparticle tracking analysis on polydisperse macromolecule assembly. Sci Rep 9(1):2639. https://doi.org/10.1038/s41598-019-38915-x Hole P, Sillence K, Hannell C, Maguire CM, Roesslein M, Suarez G, Capracotta S, Magdolenova Z, Horev-Azaria L, Dybowska A, Cooke L, Haase A, Contal S, Mano S, Vennemann A, Sauvain JJ, Staunton KC, Anguissola S, Luch A, Wick P (2013) Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA). J Nanopart Res 15:2101. https://doi.org/10.1007/s11051-013-2101-8 El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ (2012) Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 7(12):2112–2126. https://doi.org/10.1038/nprot.2012.131 Carr B, Wright M (2013) Nanoparticle tracking analysis: a review of applications and usage 2010–2012. Nanosight Weber A, Wehmeyer JC, Schmidt V, Lichtenberg A, Akhyari P (2019) Rapid fluorescence-based characterization of single extracellular vesicles in human blood with nanoparticle-tracking analysis. J Vis Exp. https://doi.org/10.3791/58731 Dehghani M, Gulvin SM, Flax J, Gaborski TR (2020) Systematic evaluation of PKH labelling on extracellular vesicle size by nanoparticle tracking analysis. Sci Rep 10(1):9533. https://doi.org/10.1038/s41598-020-66434-7 Szatanek R, Baj-Krzyworzeka M, Zimoch J, Lekka M, Siedlar M, Baran J (2017) The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci. https://doi.org/10.3390/ijms18061153 Dragovic RA, Collett GP, Hole P, Ferguson DJ, Redman CW, Sargent IL, Tannetta DS (2015) Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence nanoparticle tracking analysis. Methods 87:64–74. https://doi.org/10.1016/j.ymeth.2015.03.028 Cho S, Yi J, Kwon Y, Kang H, Han C, Park J (2021) Multifluorescence single extracellular vesicle analysis by time-sequential illumination and tracking. ACS Nano. https://doi.org/10.1021/acsnano.1c02556 Melling GE, Conlon R, Pantazi P, Dellar ER, Samuel P, Baena-Lopez LA, Simpson JC, Carter DRF (2022) Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods. Sci Rep 12(1):262. https://doi.org/10.1038/s41598-021-04225-4 Simonsen JB (2019) Pitfalls associated with lipophilic fluorophore staining of extracellular vesicles for uptake studies. J Extracell Vesicles 8(1):1582237. https://doi.org/10.1080/20013078.2019.1582237 Kerker M, Chew H, McNulty PJ, Kratohvil JP, Cooke DD, Sculley M, Lee MP (1979) Light scattering and fluorescence by small particles having internal structure. J Histochem Cytochem 27(1):250–263. https://doi.org/10.1177/27.1.438501 Tian Y, Ma L, Gong M, Su G, Zhu S, Zhang W, Wang S, Li Z, Chen C, Li L, Wu L, Yan X (2018) Protein profiling and sizing of extracellular vesicles from colorectal cancer patients via flow cytometry. ACS Nano 12(1):671–680. https://doi.org/10.1021/acsnano.7b07782 Nolte-’t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine 8(5):712–720. https://doi.org/10.1016/j.nano.2011.09.006 Ma L, Zhu S, Tian Y, Zhang W, Wang S, Chen C, Wu L, Yan X (2016) Label-free analysis of single viruses with a resolution comparable to that of electron microscopy and the throughput of flow cytometry. Angew Chem Int Ed Engl 55(35):10239–10243. https://doi.org/10.1002/anie.201603007 van der Vlist EJ, Nolte-’t Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc 7(7):1311–1326. https://doi.org/10.1038/nprot.2012.065 Zhu S, Ma L, Wang S, Chen C, Zhang W, Yang L, Hang W, Nolan JP, Wu L, Yan X (2014) Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano 8(10):10998–11006. https://doi.org/10.1021/nn505162u Shapiro HM (2005) Practical flow cytometry. Wiley, Oxford. https://doi.org/10.1002/0471722731 Steen HB (2004) Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry 57A(2):94–99. https://doi.org/10.1002/cyto.a.10115 Libregts S, Arkesteijn GJA, Nemeth A, Nolte-’t Hoen ENM, Wauben MHM (2018) Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest. J Thromb Haemost 16(7):1423–1436. https://doi.org/10.1111/jth.14154 Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC (2017) Labeling extracellular vesicles for nanoscale flow cytometry. Sci Rep 7(1):1878. https://doi.org/10.1038/s41598-017-01731-2 Groot Kormelink T, Arkesteijn GJ, Nauwelaers FA, van den Engh G, Nolte-’t Hoen EN, Wauben MH (2016) Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Cytometry A 89(2):135–147. https://doi.org/10.1002/cyto.a.22644 Arraud N, Gounou C, Linares R, Brisson AR (2015) A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles. J Thromb Haemost 13(2):237–247. https://doi.org/10.1111/jth.12767 Ter-Ovanesyan D, Kowal EJK, Regev A, Church GM, Cocucci E (2017) Imaging of isolated extracellular vesicles using fluorescence microscopy. Methods Mol Biol 1660:233–241. https://doi.org/10.1007/978-1-4939-7253-1_19 Silva AM, Lazaro-Ibanez E, Gunnarsson A, Dhande A, Daaboul G, Peacock B, Osteikoetxea X, Salmond N, Friis KP, Shatnyeva O, Dekker N (2021) Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution. J Extracell Vesicles 10(10):e12130. https://doi.org/10.1002/jev2.12130 Han C, Kang H, Yi J, Kang M, Lee H, Kwon Y, Jung J, Lee J, Park J (2021) Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles. J Extracell Vesicles 10(3):e12047. https://doi.org/10.1002/jev2.12047 Lee J, Kwon Y, Jung J, Shin H, Park J (2021) Immunostaining extracellular vesicles based on an aqueous two-phase system: for analysis of tetraspanins. ACS Appl Bio Mater 4(4):3294–3303. https://doi.org/10.1021/acsabm.0c01625 Olsson T, Zhdanov VP, Höök F (2015) Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: theory and experiment. J Appl Phys 118(6):064702. https://doi.org/10.1063/1.4928083 Shen L-M, Quan L, Liu J (2018) Tracking exosomes in vitro and in vivo to elucidate their physiological functions: implications for diagnostic and therapeutic nanocarriers. ACS Appl Nano Mater 1(6):2438–2448. https://doi.org/10.1021/acsanm.8b00601 Ewers H, Smith AE, Sbalzarini IF, Lilie H, Koumoutsakos P, Helenius A (2005) Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes. Proc Natl Acad Sci U S A 102(42):15110–15115. https://doi.org/10.1073/pnas.0504407102 Gul B, Ashraf S, Khan S, Nisar H, Ahmad I (2021) Cell refractive index: models, insights, applications and future perspectives. Photodiagnosis Photodyn Ther 33:102096. https://doi.org/10.1016/j.pdpdt.2020.102096 Liu PY, Chin LK, Ser W, Chen HF, Hsieh CM, Lee CH, Sung KB, Ayi TC, Yap PH, Liedberg B, Wang K, Bourouina T, Leprince-Wang Y (2016) Cell refractive index for cell biology and disease diagnosis: past, present and future. Lab Chip 16(4):634–644. https://doi.org/10.1039/c5lc01445j Hoshino D, Kirkbride KC, Costello K, Clark ES, Sinha S, Grega-Larson N, Tyska MJ, Weaver AM (2013) Exosome secretion is enhanced by invadopodia and drives invasive behavior. Cell Rep 5(5):1159–1168. https://doi.org/10.1016/j.celrep.2013.10.050 He D, Wang H, Ho SL, Chan HN, Hai L, He X, Wang K, Li HW (2019) Total internal reflection-based single-vesicle in situ quantitative and stoichiometric analysis of tumor-derived exosomal microRNAs for diagnosis and treatment monitoring. Theranostics 9(15):4494–4507. https://doi.org/10.7150/thno.33683 Jiang Y, Andronico LA, Jung SR, Chen H, Fujimoto B, Vojtech L, Chiu DT (2021) High-throughput counting and superresolution mapping of tetraspanins on exosomes using a single-molecule sensitive flow technique and transistor-like semiconducting polymer dots. Angew Chem Int Ed Engl 60(24):13470–13475. https://doi.org/10.1002/anie.202103282 Raschke G, Brogl S, Susha AS, Rogach AL, Klar TA, Feldmann J, Fieres B, Petkov N, Bein T, Nichtl A, Kürzinger K (2004) Gold nanoshells improve single nanoparticle molecular sensors. Nano Lett 4(10):1853–1857. https://doi.org/10.1021/nl049038q Hartland GV (2011) Optical studies of dynamics in noble metal nanostructures. Chem Rev 111(6):3858–3887. https://doi.org/10.1021/cr1002547 Schultz S, Smith DR, Mock JJ, Schultz DA (2000) Single-target molecule detection with nonbleaching multicolor optical immunolabels. Proc Natl Acad Sci 97(3):996–1001. https://doi.org/10.1073/pnas.97.3.996 Li T, Wu X, Liu F, Li N (2017) Analytical methods based on the light-scattering of plasmonic nanoparticles at the single particle level with dark-field microscopy imaging. Analyst 142(2):248–256. https://doi.org/10.1039/c6an02384c Ekiz-Kanik F, Sevenler DD, Ünlü NL, Chiari M, Ünlü MS (2017) Surface chemistry and morphology in single particle optical imaging. Nanophotonics 6(4):713–730. https://doi.org/10.1515/nanoph-2016-0184 Enoki S, Iino R, Morone N, Kaihatsu K, Sakakihara S, Kato N, Noji H (2012) Label-free single-particle imaging of the influenza virus by objective-type total internal reflection dark-field microscopy. PLoS ONE 7(11):e49208. https://doi.org/10.1371/journal.pone.0049208 Akagi T, Kato K, Hanamura N, Kobayashi M, Ichiki T (2014) Evaluation of desialylation effect on zeta potential of extracellular vesicles secreted from human prostate cancer cells by on-chip microcapillary electrophoresis. Jpn J Appl Phys. https://doi.org/10.7567/jjap.53.06jl01 Sharma S, Rasool HI, Palanisamy V, Mathisen C, Schmidt M, Wong DT, Gimzewski JK (2010) Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 4(4):1921–1926. https://doi.org/10.1021/nn901824n Noble JM, Roberts LM, Vidavsky N, Chiou AE, Fischbach C, Paszek MJ, Estroff LA, Kourkoutis LF (2020) Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles. J Struct Biol 210(1):107474. https://doi.org/10.1016/j.jsb.2020.107474 Rupert DLM, Claudio V, Lasser C, Bally M (2017) Methods for the physical characterization and quantification of extracellular vesicles in biological samples. Biochim Biophys Acta Gen Subj 1861(1 Pt A):3164–3179. https://doi.org/10.1016/j.bbagen.2016.07.028 Arraud N, Linares R, Tan S, Gounou C, Pasquet JM, Mornet S, Brisson AR (2014) Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost 12(5):614–627. https://doi.org/10.1111/jth.12554 Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P, Haselman U, Santarella-Mellwig R, Habermann A, Hoppe S, Kallis S, Walther P, Antony C, Krijnse-Locker J, Bartenschlager R (2012) Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 8(12):e1003056. https://doi.org/10.1371/journal.ppat.1003056 Choi H, Mun JY (2017) Structural analysis of exosomes using different types of electron microscopy. Appl Microsc 47(3):171–175. https://doi.org/10.9729/am.2017.47.3.171 Emelyanov A, Shtam T, Kamyshinsky R, Garaeva L, Verlov N, Miliukhina I, Kudrevatykh A, Gavrilov G, Zabrodskaya Y, Pchelina S, Konevega A (2020) Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid. PLoS ONE 15(1):e0227949. https://doi.org/10.1371/journal.pone.0227949 Kim DH, Kim H, Choi YJ, Kim SY, Lee JE, Sung KJ, Sung YH, Pack CG, Jung MK, Han B, Kim K, Kim WS, Nam SJ, Choi CM, Yun M, Lee JC, Rho JK (2019) Exosomal PD-L1 promotes tumor growth through immune escape in non-small cell lung cancer. Exp Mol Med 51(8):1–13. https://doi.org/10.1038/s12276-019-0295-2 Baena V, Schalek RL, Lichtman JW, Terasaki M (2019) Serial-section electron microscopy using automated tape-collecting ultramicrotome (ATUM). Methods Cell Biol 152:41–67. https://doi.org/10.1016/bs.mcb.2019.04.004 Cretoiu D, Gherghiceanu M, Hummel E, Zimmermann H, Simionescu O, Popescu LM (2015) FIB-SEM tomography of human skin telocytes and their extracellular vesicles. J Cell Mol Med 19(4):714–722. https://doi.org/10.1111/jcmm.12578 Peddie CJ, Collinson LM (2014) Exploring the third dimension: volume electron microscopy comes of age. Micron 61:9–19. https://doi.org/10.1016/j.micron.2014.01.009 Briggman KL, Bock DD (2012) Volume electron microscopy for neuronal circuit reconstruction. Curr Opin Neurobiol 22(1):154–161. https://doi.org/10.1016/j.conb.2011.10.022 Li MI, Xu X, Xi N, Wang W, Xing X, Liu L (2021) Multiparametric atomic force microscopy imaging of single native exosomes. Acta Biochim Biophys Sin (Shanghai) 53(3):385–388. https://doi.org/10.1093/abbs/gmaa172 Vorselen D, Piontek MC, Roos WH, Wuite GJL (2020) Mechanical characterization of liposomes and extracellular vesicles, a protocol. Front Mol Biosci 7:139. https://doi.org/10.3389/fmolb.2020.00139 Ridolfi A, Brucale M, Montis C, Caselli L, Paolini L, Borup A, Boysen AT, Loria F, van Herwijnen MJC, Kleinjan M, Nejsum P, Zarovni N, Wauben MHM, Berti D, Bergese P, Valle F (2020) AFM-based high-throughput nanomechanical screening of single extracellular vesicles. Anal Chem 92(15):10274–10282. https://doi.org/10.1021/acs.analchem.9b05716 Yokota S, Kuramochi H, Okubo K, Iwaya A, Tsuchiya S, Ichiki T (2019) Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes. PLoS ONE 14(10):e0224091. https://doi.org/10.1371/journal.pone.0224091 Bagrov DV, Senkovenko AM, Nikishin II, Skryabin GO, Kopnin PB, Tchevkina EM (2021) Application of AFM, TEM, and NTA for characterization of exosomes produced by placenta-derived mesenchymal cells. J Phys Conf Ser 1942(1):012013. https://doi.org/10.1088/1742-6596/1942/1/012013 Yurtsever A, Yoshida T, Badami Behjat A, Araki Y, Hanayama R, Fukuma T (2021) Structural and mechanical characteristics of exosomes from osteosarcoma cells explored by 3D-atomic force microscopy. Nanoscale 13(13):6661–6677. https://doi.org/10.1039/d0nr09178b Rawicz W, Olbrich K, McIntosh T, Needham D, Evans E (2000) Effect of chain length and unsaturation on elasticity of lipid bilayers. Biophys J 79(1):328–339. https://doi.org/10.1016/s0006-3495(00)76295-3 Dimova R (2014) Recent developments in the field of bending rigidity measurements on membranes. Adv Colloid Interface Sci 208:225–234. https://doi.org/10.1016/j.cis.2014.03.003 Sorkin R, Huisjes R, Boskovic F, Vorselen D, Pignatelli S, Ofir-Birin Y, Freitas Leal JK, Schiller J, Mullick D, Roos WH, Bosman G, Regev-Rudzki N, Schiffelers RM, Wuite GJL (2018) Nanomechanics of extracellular vesicles reveals vesiculation pathways. Small 14(39):e1801650. https://doi.org/10.1002/smll.201801650 LeClaire M, Gimzewski J, Sharma S (2020) A review of the biomechanical properties of single extracellular vesicles. Nano Sel 2(1):1–15. https://doi.org/10.1002/nano.202000129 Morshed A, Karawdeniya BI, Bandara Y, Kim MJ, Dutta P (2020) Mechanical characterization of vesicles and cells: a review. Electrophoresis 41(7–8):449–470. https://doi.org/10.1002/elps.201900362 Calo A, Reguera D, Oncins G, Persuy MA, Sanz G, Lobasso S, Corcelli A, Pajot-Augy E, Gomila G (2014) Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young’s modulus. Nanoscale 6(4):2275–2285. https://doi.org/10.1039/c3nr05107b Lane RE, Korbie D, Anderson W, Vaidyanathan R, Trau M (2015) Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing. Sci Rep 5:7639. https://doi.org/10.1038/srep07639 Buzas EI, Gardiner C, Lee C, Smith ZJ (2017) Single particle analysis: methods for detection of platelet extracellular vesicles in suspension (excluding flow cytometry). Platelets 28(3):249–255. https://doi.org/10.1080/09537104.2016.1260704 Blundell ELCJ, Mayne LJ, Billinge ER, Platt M (2015) Emergence of tunable resistive pulse sensing as a biosensor. Anal Methods 7(17):7055–7066. https://doi.org/10.1039/c4ay03023k Anderson W, Lane R, Korbie D, Trau M (2015) Observations of tunable resistive pulse sensing for exosome analysis: improving system sensitivity and stability. Langmuir 31(23):6577–6587. https://doi.org/10.1021/acs.langmuir.5b01402 Maas SL, Broekman ML, de Vrij J (2017) Tunable resistive pulse sensing for the characterization of extracellular vesicles. Methods mol biol (Clifton, N.J.) 1545:21–33. https://doi.org/10.1007/978-1-4939-6728-5_2 Yang L, Yamamoto T (2016) Quantification of virus particles using nanopore-based resistive-pulse sensing techniques. Front Microbiol 7:1500. https://doi.org/10.3389/fmicb.2016.01500 Vogel R, Willmott G, Kozak D, Roberts GS, Anderson W, Groenewegen L, Glossop B, Barnett A, Turner A, Trau M (2011) Quantitative sizing of nano/microparticles with a tunable elastomeric pore sensor. Anal Chem 83(9):3499–3506. https://doi.org/10.1021/ac200195n Liao C, Antaw F, Wuethrich A, Anderson W, Trau M (2020) Configurable miniaturized 3D pores for robust single-nanoparticle analysis. Small Struct. https://doi.org/10.1002/sstr.202000011 Arab T, Mallick ER, Huang Y, Dong L, Liao Z, Zhao Z, Gololobova O, Smith B, Haughey NJ, Pienta KJ, Slusher BS, Tarwater PM, Tosar JP, Zivkovic AM, Vreeland WN, Paulaitis ME, Witwer KW (2021) Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms. J Extracell Vesicles 10(6):e12079. https://doi.org/10.1002/jev2.12079 Ciardiello C, Cavallini L, Spinelli C, Yang J, Reis-Sobreiro M, de Candia P, Minciacchi VR, Di Vizio D (2016) Focus on extracellular vesicles: new frontiers of cell-to-cell communication in cancer. Int J Mol Sci 17(2):175. https://doi.org/10.3390/ijms17020175