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Structural studies of microbubbles and molecular chaperones using transmission electron microscopy
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.ORCID iD: 0000-0002-9604-0511
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ultrasound contrast agents (CAs) are typically used in clinic for perfusion studies (blood flow through a specific region) and border delineating (differentiate borders between tissue structures) during cardiac imaging. The CAs used during ultrasound imaging usually consist of gas filled microbubbles (MBs) (diameter 1-5 μm) that are injected intravenously into the circulatory system. This thesis partially involves a novel polymer-shelled ultrasound CA that consists of air filled MBs stabilized by a polyvinyl alcohol (PVA) shell. These MBs could be coupled with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as a combined CA for ultrasound and magnetic resonance imaging. The first three papers (Paper A-C) in this thesis investigate the structural characteristic and the elimination process of the CA.

In Paper A, two types (PVA Type A and PVA Type B) of the novel CA were analyzed using transmission electron microscopy (TEM) images of thin sectioned MBs. The images demonstrated that the SPIONs were either attached to the PVA shell surface (PVA Type A) or embedded in the shell (PVA Type B). The average shell thickness of the MBs was determined in Paper B by introducing a model that calculated the shell thickness from TEM images of cross-sectioned MBs. The shell thickness of PVA Type A was determined to 651 nm, whereas the shell thickness of PVA Type B was calculated to 637 nm. In Paper C, a prolonged blood elimination time was obtained for PVA-shelled MBs compared to the lipid-shelled CA SonoVue used in clinic. In addition, TEM analyzed tissue sections showed that the PVA-shelled MBs were recognized by the macrophage system. However, structurally intact MBs were still found in the circulation 24 h post injection. These studies illustrate that the PVA-shelled MBs are stable and offer large chemical variability, which make them suitable as CA for multimodal imaging.

This thesis also involves studies (Paper D-E) of the molecular chaperones (Hsp21 and DNAJB6). The small heat shock protein Hsp21 effectively protects other proteins from unfolding and aggregation during stress. This chaperone ability requires oligomerization of the protein. In Paper D, cryo-electron microscopy together with complementary structural methods, obtained a structure model which showed that the Hsp21 dodecamer (12-mer) is kept together by paired C-terminal interactions.The human protein DNAJB6 functions as a very efficient suppressor of polyglutamine (polyQ) and amyloid-β42 (Aβ42) aggregation. Aggregation of these peptides are associated with development of Huntington’s (polyQ) and Alzheimer’s (Aβ42) disease. In Paper E, a reconstructed map of this highly dynamic protein is presented, showing an oligomer with two-fold symmetry, indicating that the oligomers are assembled by two subunits.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. , xiii, 58 s p.
Series
TRITA-STH : report, ISSN 1653-3836 ; 2016:3
Keyword [en]
Transmission electron microscopy, Contrast agent, Microbubble, Polyvinyl alcohol, Single particle analysis, Heat shock protein, Molecular chaperone
National Category
Structural Biology
Research subject
Technology and Health
Identifiers
URN: urn:nbn:se:kth:diva-186882ISBN: 978-91-7729-020-9 (print)OAI: oai:DiVA.org:kth-186882DiVA: diva2:928863
Public defence
2016-06-08, Hörsalen plan 4 Novum, Blickagången 6, 141 57, Huddinge, 10:00 (English)
Opponent
Supervisors
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-16 Last updated: 2016-05-27Bibliographically approved
List of papers
1. Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging
Open this publication in new window or tab >>Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging
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2012 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 13, no 5, 1390-1399 p.Article in journal (Refereed) Published
Abstract [en]

Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

Keyword
Ultrasound Contrast Agents, Iron-Oxide Nanoparticles, Active Polymeric Microbubbles, Coated Microbubbles, Acoustic Properties, Induced Fracture, In-Vitro, Part I, Delivery, Shell
National Category
Medical Engineering
Identifiers
urn:nbn:se:kth:diva-96726 (URN)10.1021/bm300099f (DOI)000303951600019 ()2-s2.0-84861134310 (Scopus ID)
Note

QC 20150626

Available from: 2012-06-12 Created: 2012-06-11 Last updated: 2017-12-07Bibliographically approved
2. Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy
Open this publication in new window or tab >>Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy
2016 (English)In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 85, 39-43 p.Article in journal (Refereed) Published
Abstract [en]

Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.

Place, publisher, year, edition, pages
Elsevier, 2016
Keyword
Shell thickness, Contrast agent, Transmission electron microscopy
National Category
Medical Engineering
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-186876 (URN)10.1016/j.micron.2016.03.009 (DOI)000376217400006 ()27077316 (PubMedID)2-s2.0-84962835270 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 245572
Note

QC 20160516

Available from: 2016-05-16 Created: 2016-05-16 Last updated: 2017-11-30Bibliographically approved
3. Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy
Open this publication in new window or tab >>Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy
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2015 (English)In: Biomedical Spectroscopy and Imaging, ISSN 2212-8794, Vol. 4, no 1, 81-93 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: A novel polymer-shelled contrast agent (CA) with multimodal imaging and target specific potential was developed recently and tested for its acoustical properties using different in-vitro setups.

OBJECTIVE: The aim of this study was to investigate the elimination of three types of the novel polymer-shelled CA, one unmodified and two shell modified versions, in rats.

METHODS: The blood elimination time was estimated by measuring the image intensity, from ultrasound images of the common carotid artery, over time after a bolus injection of the three types of the novel CA. The commercially available CA SonoVue was used as a reference. The subcellular localization of the three CAs was investigated using transmission electron microscopy.

RESULTS: The ultrasound measurements indicated a blood half-life of 17–85 s for the different types of the novel CA, which was significant longer than the blood half-life time for SonoVue. Additionally, CAs were exclusively found in the circulatory system, either taken up by, or found in the vicinity of macrophages.

CONCLUSIONS: Compared to the commercially available CA SonoVue, the blood circulation times for the three types of the novel polymer-shelled CA were prolonged. Moreover, macrophages were suggested to be responsible for the elimination of the CA.

Keyword
Contrast agent, polymer, subcellular localization, transmission electron microscope, ultrasound imaging
National Category
Medical Engineering
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-160905 (URN)10.3233/BSI-140099 (DOI)
Funder
EU, FP7, Seventh Framework Programme, 245572
Note

QC 20150401

Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-08-15Bibliographically approved
4. Structure model obtained by homology modelling and cryo-EM for the Hsp21 dodecamer and evaluation of the importance of oligomerization for chaperone activity
Open this publication in new window or tab >>Structure model obtained by homology modelling and cryo-EM for the Hsp21 dodecamer and evaluation of the importance of oligomerization for chaperone activity
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-187065 (URN)
Note

QC 20160623

Available from: 2016-05-16 Created: 2016-05-16 Last updated: 2016-06-23Bibliographically approved
5. Structural information on the oligomeric human molecular chaperone DNAJB6
Open this publication in new window or tab >>Structural information on the oligomeric human molecular chaperone DNAJB6
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-187066 (URN)
Note

QC 20160608

Available from: 2016-05-16 Created: 2016-05-16 Last updated: 2016-06-08Bibliographically approved

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