The search for new therapeutic molecules has brought the medicinal weapons “arsenal” to a mature stage where a drug is available for many common diseases. However, an ample spectrum of active molecules alone does not guarantee optimal therapeutic efficacy as other relevant factors come into play in modern therapies such as drug diversity, bioavailability and release modalities. For the regulation of such factors the environment in which the drug molecule is embedded has an active role and the old fashioned “excipient” is now replaced by a complex, multifunctional device partly or totally based on soft condensed matter. Such systems display unique features allowing controlled delivery of the drug and therefore being integral part of the final drug design. This is becoming a widely accepted new paradigm of the “drug” as a system whose efficacy enhancement is obtained with the concerted action of a chemical compound and of the controlled delivery system. Moreover, emerging therapies such as gene therapy cannot be conceived without the support of a soft matter delivery device as the functional integrity of the macromolecular drug relies on the properties of the embedding support. This project introduces a new class of micro/nano devices providing integrated diagnostic and therapeutic functionalities.

Ultrasound imaging is probably the most used approach for the fast diagnosis and monitoring of cardiovascular disease and for countless other routine medical actions. Convenience and versatility are established assets of this non-invasive imaging technique. The ultrasound imaging technique can be greatly improved by the use of contrast agents to enhance the signal of the area of interest (e.g. cardiac or liver tissue) relative to the background. Generally these devices are injected systemically and function to passively (non-specifically) enhance the ultrasound echo. Presently they are used  in the diagnosis of disease in the tissue being imaged.

The S.I.G.H.T. project starts from this background and aims to formulate new medical ultrasound-active devices with novel and improved properties to extend the concepts of the Molecular Imaging, (such as high resolution, affordable diagnostic for in vivo biological processes) to the next-generation of ultrasound contrast agents. Moreover, in addition to improving the diagnostic properties, this project will add a larger spectrum of functionalities to the newly formulated devices, including a therapeutic capability. Therefore the activities of S.I.G.H.T. will concern the fabrication of multifunctional devices suited for diagnosis with molecular imaging quality as well as for therapeutic treatment. This feature will facilitate the use of UCAs in much broader application fields. Table 1 summarizes the correlation between the functional and structural properties for the new concept of ultrasonically active contrast agents.

Table 1. Ideal properties of a UCA incorporating the new concept of diagnostic and therapeutic functionalities

Commercially available UCAs have several drawbacks to the general use, including high variability of the dimensions and short lifetime, which are overcome in the new design of ultrasound agent proposed in S.I.G.H.T. Injectability, ultrasound scattering efficiency and biocompatibility are functional issues to be improved in the new UCAs by controlling the corresponding structural properties. Drug payload and drug delivery are completely new features not present in the currently available products based on micro-bubbles. The addition of these properties will change the nature and functions of the new UCAs. S.IG.H.T envisages the replacement of phospholipid microbubbles with polymeric materials (micro-balloons and microcapsules) which offer a longer average life-time in vivo and better chemical and physical versatility for attaching different kinds of molecules. For this reason the project shifts from micro-bubbles to micro-balloons and micro-capsules.

The overall actions of S.I.G.H.T. are distributed into 9 Research Workpackages (WP) and one WP that dedicated to management issues:

WP  0 - Project Management
WP  1 - Synthesis, functionalization and characterisation of polymeric micro-balloons
WP  2 -  Microcapsule synthesis, functionalization and characterization
WP  3 -  Mechanical properties of micro-balloons and microcapsules
WP  4 -  Microcapsule and micro-balloon arrays
WP 5 - Bio-interfaces: Bioadhesion properties and biocompatibility of polymeric micro-balloons and microcapsules
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

First year S.I.G.H.T. activities were focused towards:

1. the formulation and validation of the protocols for micro-balloons (MBs) and microcapsules (MCs) production,
2. characterization of MBs average size and MBs size distribution,
3. acoustic behaviour of MBs,
4. preliminary mechanic behaviour of MBs,
5. surface functionalization.
6. biointerfacial behaviour of MBs with plasma.

The achieved results are essentially in agreement with the forecast work plan.

Within WP1, a set of micro-balloon samples have been prepared under different conditions of pH and temperature. Functionalization of micro-balloon surfaces with L-lysine and the tripeptide RGD was successfully carried out employing residual reactive groups on the surface, i.e. aldehydes, by Schiff base formation and reductive amination. Also a protocol for microbubbles → water filled microcapsules conversion was devised for the biointerfacial studies (carried out within the framework of WP 5). Synthetic strategies will be developed further on the basis of the results achieved in the other WP activities. Along with these objectives, the SIGHT work plan is based on the acquisition by all partners of the expertise and know-how concerning the handling and the fundamental characteristics of the microdevices. Within the framework of WP1 activity, samples with different sizes and shell thickness have been provided to all the partners involved in the first year activities. Networking, forum activity on the SIGHT website, and email exchange have supported the discussions among the different groups concerning the handling of polymer micro-balloons. As a result of the first year activity, interested groups are now in the position to reproduce the micro-balloon preparation and to obtain similar characteristics. This is considered an important step toward the development of future research activities in the program.
WP2 activity was focussed on layer-by-layer microcapsule fabrication for developing diagnostic microarrays (carried out within the framework of WP4). Preliminary coating of micro-balloons using LbL technology was also examined. Characterization of microparticles by atomic force microscopy and laser scanning confocal microscopy has been carried out in this WP.

Acoustic and mechanical properties of micro-balloons were studied in WP3. Interesting features of micro-balloons  emerged in this study. A threshold pressure, Pthr, due to insonification was detected, above which the micro-balloons rupture. The linear behaviour of the micro-balloons  acoustic properties below this threshold was used to assess the viscoelastic properties of the polymer shell and the imaging effectiveness, whereas the region above Pthr is suitable for the release of drugs in view of a therapeutic use of the device. AFM studies focussed on the mechanical properties of micro-balloons, based on force-deformation experiments have enabled an assessment of the force-induced burst process.

Microarray fabrication, which is mainly treated in WP4, is an important issue for using microcapsules and micro-balloons in the design of diagnostic devices. Adhesion to charged flat substrates with positive or negative charges has been worked out for microcapsules and micro-balloons. The adhesiveness of microcapsules has been assessed as suitable for further development of microarrays, whereas tests on micro-balloons has indicated poor adhesion on the same supports. Use of the novel scanning X-ray transmission microscopy, SXTM, not planned in the work program, showed the suitability of this technique for high resolution imaging and for determination of localized chemical composition.

Biocompatibility tests were performed in WP5 on micro-balloons converted into water-filled microcapsules (see WP1). This strategy was adopted in order to guarantee contact between the micro-balloons and cells, maintaining the chemical characteristics of the micro-balloon surfaces. Primary immunoresponse was one focus of this WP, as the theranostic action of micro-balloons is based on their injectability. Fibroblast and macrophage cell lines were selected as models and were exposed to polymer shelled water-filled microcapsules. Viability and proliferation tests allowed assessment of the biocompatibility of the microcapsules and an in vitro evaluation of the primary immunoresponse. This study was also carried out on flat membranes.

Proteomic analysis was performed to identify the serum proteins associated with the micro-balloons, in order to characterise the corona which is responsible for any biological responses elicited. Quite interesting and unusual features emerged in this study as a remarkable selectivity for red blood-cell associated proteins was noted. The implications of this finding have still to be evaluated. A preliminary assessment of the effects of insonification on normal and tumour cells with or without microballoons was performed, using cell viability and proliferation as endpoints.

According to the SIGHT work plan, investigation of drug release performance upon insonification with endothelial cells and for clot disruption (WP6) and on the release performance of therapeutic molecules upon insonification with human hepatocellular carcinoma and colon cancer cells (WP7) will start from the second year. Therefore preliminary achievements on these activities will be reported only at the end of the second year.

The evaluation of the state-of-art of image processing approaches, with a focus on selected signal processing algorithms suitable for use with ultrasound contrast agents with novel acoustic properties, has been initiated within the framework of WP8.

On the basis of the achievements generated during SIGHTs first year of activity, a few considerations are in order:

1. the Consortium is working in a concerted way with an efficient networking activity to broaden the expertise on the microdevices (micro-balloons and microcapsules) within the partner institutes.
2. The work plan objectives have been mainly matched, although several issues require continued research activities in order to finalize, confirm and interpret the results obtained to date.

Functionalization and surface modification of both capsules and balloons will continue, as well as investigation of the implications of these activities in several issues of the project, such as microarray constitution, biointerfacial features, and acoustic, mechanical and image processing properties. An unusual and new ability of the micro-balloons surface to trap specific proteins could offer routes to other unforeseen applications. On the methodological side, an alternative technique, not included in the workplan, STXM has provided new insights in the study of micro-balloons, and concerted use of this approach will be developed during the second year of activities.

It is quite easy to predict a sustained knowledge increase relating to these devices within SIGHT, with direct outputs including publication of papers, training of new scientists and potentially formulation of dedicated devices for the biomedical applications.

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