The S.I.G.H.T. project aims to formulate next-generation ultrasound contrast agents with novel functionalities and improved properties such as higher resolution affordable diagnostics for in vivo imaging of biological processes. In addition to improving the imaging and diagnostic properties, this project will add a larger spectrum of functionalities to the newly formulated devices, including a therapeutic capability. Significant progress was made in the first year of activity, as summarised in the previous Publishable Executive Summary.  

According to the project workplan, the second year activity is devoted to the accomplishment of intermediate objectives concerning the structure-properties relationships (shell structure – acoustic and mechanical behaviour), microballoon surface functionalization for targeting and drug loading, preliminary assessment of the imaging properties of the microballoons, and proteomics assessment of the variously conjugated surfaces. During the second year the project has changed some of the major objectives, specifically those related to use of liquid-filled layer-by-layer microcapsules due to their unfavourable performance with respect to ultrasound scattering efficiency. This challenge, combined with the exit of Medtronic from the project, has provided the consortium with an opportunity to re-consider the work plan and to develop a new strategy to replace the microcapsules. The new system, layer-by-layer microballoons combines the scattering properties of microballoons with the advantages of the layer-by-layer technology for surface modification. A close collaboration between partner 2 (UNITV) and partner 9 (CAPS) achieved in a very short time an assessment of the new system, enabling the consortium to adapt the project without compromising the main objectives of the project.
The overall activities of the S.I.G.H.T. project are distributed across the following workpackages:
WP 0 - Project Management
WP 1 - Synthesis, functionalization and characterisation of polymeric microballoons
WP 2 - Microcapsule synthesis, functionalization and characterization (now re-directed towards layer-by-layer coated microballoons)
WP 3 - Mechanical properties of microballoons and microcapsules (LbL-coated microballoons)
WP 4 - Microcapsule and microballoon arrays
WP 5 - Bio-interfaces: Bioadhesion properties and biocompatibility of polymeric microballoons
WP 6 - Drug release performance upon insonification in endothelial cells and for clot
disruption.
WP 7 - Release performance of therapeutic molecules upon insonification in human
hepatocellular carcinoma and colon cancer cells.
WP 8 - Image Processing and Reconstruction
WP 9 - Dissemination and Exploitation Promotion
Note that for the remaining time period of the project, LbL-coated microballoons will replace the microcapsules in all WPs.
 
During the 2nd year of the S.I.G.H.T. project the research activities were focused towards:
- Strategy assessment for loading of microballoons with therapeutic gases, including NO.
- Strategy assessment for targeting and drug loading of microballoons
- Assessment of the microballoon shell structure and water content
- Assessment of the acoustic behaviour of surface modified microballoons
- Assessment of the mechanical behaviour of microballoons
- Assessment of the impact of ultrasound assisted drug release on cancer cells.
- Biointerfacial behaviour of microballoons with human plasma.
- Bioadhesion of surface engineered microballoons.

The results achieved during the second period of activitiy are essentially in agreement with the workplan forecast. The main achievements from this period can be summarised as follows:

• A range of fucntionalised microballoons have been prepared and are being characterised in terms of the acoustic, mechanical, and adhesive properties, and their therapeutic potential.
• Functionalisations include hyaluronic acid (HA), a natural polysaccharide known to be the ligand of CD44 receptor in tumour cell, and RGD, a tripeptide (arg-gly-asp) that binds integrins, chitosan to introduce charges onto the surface, and cyclodextrin to facilitate drug conjugation.
• The chitosan-functionalisation of microballoons was a real breakthrough allowing the layer-by-layer technology to be applied to microballoons, as shown in Figure 1.

Sulforhodamine (-) 11mStd	Rhodamine-B (+/-) 8mStd	Rhodamine-6G (+) 69mStd
Figure 1 Confocal microscopy images of differently charged dyes immobilized on the surface of chitosan-conjugated microballoons (image size 40 µm x 40 µm) illustrating the success of the conjugations strategy and the layer-by-layer coating of microballoons.

• Investigation of the acoustic and mechanical properties of the MBs have shown that fatigue or accumulation of damage within the shell plays an important role in fracture of the microballoons, which has important implications for drug release from the shells.
• STXM enabled characterisation of the microballoon shell thickness and composition, and resulted in the development of a deformation model based on two distinct deformation states – non-squeezed and squeezed, as shown in Figure 2.  Experimental data can be explained using this model.

	Figure 2 – STXM image (left) and radial transmission profile (center, dots) of Microballoon B and schematic of the deformed model (right) used to explain the radial profiles (center, line).

Hyaluronic acid was conjugated to MBs to improve their bioadhesivenss, especially towards cells with upregulated CD44 receptros, such as occurs in cancerous cells. Uptake studies of the HA-modified Microballooons show that the HA modification does indeed result in an enhanced interaction with both macrophages and fibroblasts, as shown in Figure 3.

Figure 3 Confocal microscopy images of NIH3T3 mouse fibroblasts (a) and RAW264,7 mouse macrophages (b) incubated with FITC conjugated HA-MBs. Cells are stained with Dil stain (red color).

• Several approaches for drug-loading of the MBS have been developed, and release studies have shown that drugs (such as doxycycline, doxorubicin) and therapeutic gases (such as NO) released from MBs retain their activity and efficacy, showing proof of principle of the theranostic ultrasound contrast devices.
• Cytotoxicity tests have revealed that the MBs and functionalised MBs show little toxicity or DNA damage to a range of cells. Advanced assessment of the potential biological impacts of the MBs is underway, involving proteomic assessment of the adsorbed protein corona.
• Detailed modelling of the non-linear signal propagated by the MBs is also underway, in order to assess the behaviour of microballoon populations in relation to existing ultrasound devices in use in hospitals, in order to optimise the backscatter and contrast.

The project is carried out by a pan-European consortium:

• Consorzio Roma Ricerche - CRR (Italy)
• Università degli Studi di Roma Tor Vergata  - UNITV (Italy)
• Kungliga Tekniska Hogskolan  - KTH (Sweden)
• University College Dublin / National University of Ireland, Dublin - UCD (Ireland)-
• Karolinska Institutet - KI (Sweden)
• Fondazione IRCCS Istituto Nazionale dei Tumori - INT (Italy)
• Medtronic Bakken Research Center* -MDT (The Netherlands )
• Capsulution NanoScience AG  - CAPS (Germany)
• Universität Bayreuth - UBT (Germany)
• ESAOTE Spa (Italy)

* Withdrew from the project during the 2nd Activity Period due to internal restructuring.

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