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Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics. (Prof. Jan Linnros)ORCID iD: 0000-0002-5077-3218
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. 

First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. 

Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. 

Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2021. , p. 97
Series
TRITA-SCI-FOU ; 2021:44
Keywords [en]
extracellular vesicles, streaming current, fluorescence microscopy, atomic force microscopy, scanning electron microscopy, protein profiling, size profiling
National Category
Nano Technology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
Physics, Biological and Biomedical Physics
Identifiers
URN: urn:nbn:se:kth:diva-304800ISBN: 978-91-8040-069-5 (print)OAI: oai:DiVA.org:kth-304800DiVA, id: diva2:1611019
Public defence
2021-12-10, Room Ångdomen and via Zoom: https://kth-se.zoom.us/j/68480621469, Osquars backe 31, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2021-11-15 Created: 2021-11-12 Last updated: 2022-09-21Bibliographically approved
List of papers
1. Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
Open this publication in new window or tab >>Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
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2019 (English)In: ACS Sensors, E-ISSN 2379-3694, Vol. 4, no 5, p. 1399-1408Article in journal (Refereed) Published
Abstract [en]

Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
extracellular vesicles, electrokinetic effect, biosensor, label-free, protein profiling, cancer
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-254037 (URN)10.1021/acssensors.9b00418 (DOI)000469410100034 ()31020844 (PubMedID)2-s2.0-85066017871 (Scopus ID)
Note

Qc 20190814

Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2024-03-18Bibliographically approved
2. Influence of molecular size and zeta potential in electrokinetic biosensing
Open this publication in new window or tab >>Influence of molecular size and zeta potential in electrokinetic biosensing
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2020 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 152, article id 112005Article in journal (Refereed) Published
Abstract [en]

Electrokinetic principles such as streaming current and streaming potential are extensively used for surface characterization. Recently, they have also been used in biosensors, resulting in enhanced sensitivity and simpler device architecture. Theoretical models regarding streaming current/potential studies of particle-covered surfaces have identified features such as the particle size, shape and surface charge to influence the electrokinetic signals and consequently, the sensitivity and effective operational regime of the biosensor. By using a set of well-characterized proteins with varying size and net surface charge, this article experimentally verifies the theoretical predictions about their influence on the sensor signal. Increasing protein size was shown to enhance the signal when their net surface charge was either opposite to that of the sensor surface, or close to zero, in agreement with the theoretical predictions. However, the effect gradually saturated as the protein size exceeded the coulomb screening length of the electrolyte. In contrast, the proteins containing the same type of charge as the surface showed little or no difference, except that the signal inverted. The magnitude of the surface charge was also shown to influence the signal. The sensitivity of the technique for protein detection varied over two orders of magnitude, depending upon the size and surface charge. Furthermore, the capacity of the electrokinetic method for direct electrical detection of various proteins, including those carrying little or no net electric charges, is demonstrated.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Biosensor, Electrokinetics, Improved sensitivity, Influence of molecular size and charge, Streaming current, Zeta potential
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-267788 (URN)10.1016/j.bios.2020.112005 (DOI)000515210600008 ()32056733 (PubMedID)2-s2.0-85077917325 (Scopus ID)
Note

QC 20200227

Available from: 2020-02-27 Created: 2020-02-27 Last updated: 2022-06-26Bibliographically approved
3. Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features
Open this publication in new window or tab >>Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features
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2021 (English)In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 17, no 14, article id 2008155Article in journal (Refereed) Published
Abstract [en]

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

Place, publisher, year, edition, pages
Wiley, 2021
Keywords
AFM, extracellular vesicles, fluorescence microscopy, mechanical properties, protein profiling, single vesicle profiling, size profiling
National Category
Basic Medicine
Identifiers
urn:nbn:se:kth:diva-293079 (URN)10.1002/smll.202008155 (DOI)000626029700001 ()33682363 (PubMedID)2-s2.0-85102149509 (Scopus ID)
Note

QC 20210420

Available from: 2021-04-20 Created: 2021-04-20 Last updated: 2024-03-18Bibliographically approved
4. Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis
Open this publication in new window or tab >>Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis
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2021 (English)In: Nanoscale Advances, E-ISSN 2516-0230, Vol. 3, no 11, p. 3053-3063Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Royal Society of Chemistry (RSC), 2021
Keywords
General Engineering, General Materials Science, General Chemistry, Atomic and Molecular Physics, and Optics, Bioengineering
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-304380 (URN)10.1039/d0na00948b (DOI)000639595400001 ()36133670 (PubMedID)2-s2.0-85107416242 (Scopus ID)
Funder
Stockholm County CouncilFamiljen Erling-Perssons StiftelseSwedish Research CouncilSwedish Cancer Society
Note

QC 20211103

Available from: 2021-11-03 Created: 2021-11-03 Last updated: 2023-09-21Bibliographically approved
5. Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients
Open this publication in new window or tab >>Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients
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2021 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 193, p. 113568-113568, article id 113568Article in journal (Refereed) Published
Keywords
Electrochemistry, Biomedical Engineering, General Medicine, Biophysics, Biotechnology
National Category
Medical Engineering
Identifiers
urn:nbn:se:kth:diva-304230 (URN)10.1016/j.bios.2021.113568 (DOI)000700077900008 ()34428672 (PubMedID)2-s2.0-85113280779 (Scopus ID)
Note

QC 20211103

Available from: 2021-10-28 Created: 2021-10-28 Last updated: 2022-06-25Bibliographically approved
6. Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor
Open this publication in new window or tab >>Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor
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2021 (English)In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 13, no 36, p. 42513-42521Article in journal (Refereed) Published
Abstract [en]

We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 x 10(6) particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2021
Keywords
streaming current, electrokinetic method, charge modulation, enhanced sensitivity, extracellular vesicles, surface proteins, lung cancer, treatment monitoring
National Category
Cancer and Oncology Cell and Molecular Biology Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-303055 (URN)10.1021/acsami.1c13192 (DOI)000697282300016 ()34473477 (PubMedID)2-s2.0-85115175404 (Scopus ID)
Note

QC 20211006

Available from: 2021-10-06 Created: 2021-10-06 Last updated: 2022-06-25Bibliographically approved

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