= The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 x 120 angstrom) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 angstrom resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.

Both diagnostic ultrasound (US) and magnetic resonance imaging (MRI) accuracy can be improved by using contrast enhancement. For US gas-filled microbubbles (MBs) or silica nanoparticles (SiNPs), and for MRI superparamagnetic or paramagnetic agents, contribute to this. However, interactions of MBs with the vascular wall and cells are not fully known for all contrast media. We studied the in vitro interactions between three types of non-targeted air-filled MBs with a polyvinyl-alcohol shell and murine macrophages or endothelial cells. The three MB types were plain MBs and two types that were labelled (internally and externally) with superparamagnetic iron oxide nanoparticles (SPIONs) for US/MRI bimodality. Cells were incubated with MBs and imaged by microscopy to evaluate uptake and adhesion. Interactions were quantified and the MB internalization was confirmed by fluorescence quenching of non-internalized MBs. Macrophages internalized each MB type within different time frames: plain MBs 6 h, externally labelled MBs 25 min and internally labelled MBs 2 h. An average of 0.14 externally labelled MBs per cell were internalized after 30 min and 1.34 after 2 h; which was 113% more MBs than the number of internalized internally labelled MBs. The macrophages engulfed these three differently modified new MBs at various rate, whereas endothelial cells did not engulf MBs. Polyvinyl-alcohol MBs are not taken up by endothelial cells. The MB uptake by macrophages is promoted by SPION labelling, in particular external such, which may be important for macrophage targeting.

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

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.

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.

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.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5. mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100. nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications, i.e. theranostics, of myocardial ischemia. We used gas filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of poly(vinyl)alcohol. In vitro acoustic tests showed a sufficient enhancement of the backscattered power (20 dB) acquired from the MBs suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1517 m/s) were comparable to those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared to clinically approved contrast agents with fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal 36 min. The application of high power ultrasound pulse (MI=1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 min in air saturated solution and from 17 to 10 min in degased solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6 to 8 hours. This is suitable time for initial diagnostics, treatment and monitoring of the therapeutic effect using single injection of the proposed multifunctional MBs.

Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.