Scientific Background

The search for new therapeutic molecules have brought the medicinal weapon “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, namely 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 a functional delivery of the drug and therefore being integral part of the final drug design. In this respect, it is becoming widely accepted a new paradigm of “drug” as a system whose efficacy enhancement is obtained with the concerted action of a chemical compound and of the controlled delivery system. Moreover, therapies assessed in recent years 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 an integrated diagnostic and therapeutic
Ultrasound imaging is probably the most used approach for fast diagnosis and monitoring of cardiovascular diseases and of countless other routine medical actions. Handiness 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 tissues) from the background. Generally these devices are injected systemically and function to passively and not specifically enhance the ultrasound echo. Presently their use aims at the diagnosis.
Ultrasound imaging is based on the back scattering efficiency of tissues or organs to be monitored. For enhanced imaging, required for more detailed investigation, there’s the employment of ultrasound contrast agents, i.e. UCAs.
The main factors acting in this process can be schematized as it follows:

Echographic Contrast Agent functioning
(for small scatterers)
 

The larger is the density and compressibility gradients between the medium and the scatterer, the better is the efficiency in scattering ultrasound waves.

From this scenario it is easy to understand that the ultrasound contrast agent must be a gas bubble. In order to stabilize the particles a shell should be provided.
The currently available echogenic micro-particles are gas-filled phospholipidic shells, i.e. micro-bubbles, characterized by a broad distribution of the particle diameters ranging from some hundreds of nanometres to tens of microns. These devices display a short life (five seconds) when injected in the systemic circulation. In order to increase the stability of the micro-particles, most commercial products have a water insoluble gas as SF6  or gaseous perfluorocarbons.
Particle dimensions are important as they should be not larger than the capillary lumen, i.e. about 5 m, and not smaller than hundreds of nanometres in order to avoid the response of the primary immunosystem, i.e. macrofagi, leucocytes.

Innovation and originality.

S.I.G.H.T. project starts from this background and aims to the formulation of new medical ultrasound-active devices with novel and improved properties for extending the concepts of the Molecular Imaging, as high resolution, affordable diagnostic for in vivo biological processes, to the next-generation of ultrasound contrast agents.
Moreover, beside the improvement of the diagnostic properties, this project will add a larger spectrum of functionalities to the new formulated devices as 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 asset will bring the use of UCAs toward much broader field of applications. Table1 summarizes the correlation between functional and structural properties of the new concept of ultrasound active agents.

Table 1. Ideal properties of a UCA new concept for diagnostic and therapeutic purposes

It is evident that the drawbacks of existing UCAs (high variability of the dimensions, short lifetime) 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 as in nowadays available products based on micro-bubbles they are not present. The addition of these properties will change nature and functions of the new UCAs according to S.I.G.H.T. design. S.IG.H.T envisages the replacement of phospholipids with polymeric materials which offer a longer average life-time and a 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.

Micro-balloons:

The use of polymers as basic materials for the fabrication of micro-balloons allows the attachment of ligand molecules and/or drug molecules on the surface. Poly (vinyl alcohol), PVA, will be used as starting material because of its biocompatibility and surfactant properties. Within the project, hybrid polymeric shell will be also studied incorporating or replacing PVA with modified biopolymers in order to enhance their surfactant properties.
The new concept of micro-balloon is schematized in Scheme 1:

The micro-balloons administered in systemic circulation, are carried by the blood stream and accumulate in the area of interest via specific ligand/receptor interaction.
Figure 1 is a laser scanning confocal micrograph on a set of poly (vinyl alcohol) micro-balloons with controlled shell thickness.

Figure 1.
A laser scanning confocal micrograph of FITC- labelled micro-balloons based on PVA (preliminary results)

The imaging will be achieved by the ultrasound scattering efficiency of the device and will be combined to the drug delivery function triggered by sonic disruption of the micro-balloon. In this way a focal and controlled time delivery of the drug will be achieved. An advantage of the focal drug delivery is a high concentration of drug released at the site where it is needed while keeping the systemic concentration of the drug as low as possible, thus reducing side effects due to bioaccumulation. Injectable micro-balloons make achievable the treatment of thrombi, tumours and tissue inflammations.
S.I.G.H.T. project will focus to clot curing and malignant tumours treatment. In clot curing the micro-balloon core will be filled with NO, a gas known to favour clot disruption. The gas release will be triggered by exploding the balloon by ultrasound irradiation and promoting the sonothrombolysis (this function can only be performed by stable NO-filled micro-balloon, acting as gas carriers in the blood stream, in the clot’s surroundings). In malignant tumours treatment the focal drug release will greatly reduce anti cancer side effects and absorption of the locally released drug will be facilitated by the transient opening of the cells called sonoporation.

Microcapsules:

S.I.G.H.T. project endorses also microcapsules as device for ultrasound diagnosis and therapy. At variance with micro-balloons, microcapsules have a solid or liquid core. Both systems are multifunctional and complementary since in the capsule case transport and delivery of liquids or solid can be carried out, although with less echogenic efficacy. Therefore, using the same type of interactions for both capsules and balloons, the efficiency of targeting/distribution of the objects in the body can be implemented by a concerted use of both devices based on polymeric materials.
In Scheme2 is described the microcapsule fabrication:

The layer-by-layer, LbL, fabrication consists in the adsorption of charged polymer molecules on solid template, followed by a deposition of a successive layer of oppositely charged polymers. By repeating this process “layer-by-layer” it is possible to obtain a micro/nano-particle with a controlled number of layers. At the end of the LbL procedure the solid core of the particle acting as deposition template is dissolved and replaced by a liquid percolating through the polymer layers.

The micrographs, Figure 2, obtained on microcapsules fabricated by LbL technology show the narrow size distribution of these micro-devices.

 
Figure 2.
Confocal micrograph of micro-capsules

The multi-functionality of these devices will be extended to ex situ applications loading the micro-devices with molecules suitable for sensoring (immunoassay and high throughput screening) by assembling micro-capsules and micro-balloons in micro-arrays, see Figure 3, capable of performing multiple testing/analysis in one step on small volumes.

Figure 3.
Array of self assembled microcapsules. Dark spots are positions of the array not yet occupied by the microcapsules (red spots).

Impact on health and quality of life.
Among the more urgent societal demands, health care is facing rapidly increasing treatment costs. Nowadays efficient health monitoring is mainly based on specificity and speed in a complex environment. This is an issue both in information technology and in medicine that can be solved by an integration process between diagnosis and therapy.
It has been evaluated that every 3 – 4 minutes a person is stroke by a heart attack and one out of four does not survive. Medical emergency treatment in this scenario is a matter of timely rescue, monitoring, diagnosis and therapeutic treatment.
Moreover, an ageing population, higher expectations for a better quality of life and changing lifestyle of European society raise the need for innovative, more efficient, less invasive and affordable health care. Early diagnosis and “smart” treatments are the two main challenges in planning the “health” demand in the near future.
Advancement of in vivo diagnostics will rely on “molecular imaging” and on minimally invasive devices. Molecular imaging aims to create highly sensitive and reliable detection agents that can also deliver and monitor therapy. The molecular imaging devices have to be designed on the basis of de novo concepts intended to make molecular process visible, quantifiable and traceable over the time. This approach has been defined “theranostics” and combines the separate disciplines of diagnosis and therapeutics.
This can only be achieved having a device able to convey a large amount of high quality images for the diagnostic step and finally a quick therapy for stabilizing the patient most of cases in the emergency car (for ischemia).
Ultrasound equipments are among the most handy, relatively economic, and patient-friendly medical apparatuses and do not require specialized users. However, enhancement of ultrasound contrast imaging is needed. An higher quality and more adequate information would provide to the physician the possibility to distinguish between normal and abnormal conditions, i.e. normal and diseased tissues. In many cases the diagnosis may face several problems: normal liver, spleen or kidney have similar acoustic properties to many tumours. Visualization of blood and blood stream within an organ is essential to distinguish normal and injured or pathological tissue.
At the present about 20% of the echographic examinations carried out on heart (echocardiography) do not provide images of adequate quality to allow the visualization of the inner border of the heart (endocardial border) for accurate diagnosis of ventricular dysfunctions.
As theranostic technologies become available physicians will be effectively empowered to bring the hospital in real time to the bedside: tissue of interest is firstly imaged using targeted contrast agents and secondly the targeting device loaded with a pharmacologically active agent can be used for therapy. Finally monitoring of the results of the therapeutic treatment over the time will be possible by sequential imaging.
The S.I.G.H.T. project is committed to the development of next-generation devices, i.e. micro-balloons and microcapsules, and of their fabrication processes representing cutting-edge advancements in diagnosis and therapeutic treatment. Improvements of the quality of life, lowering mortality rates, safety and low environmental impact are the background conditions from which the project new concept is developed.
Application of “drug-carrying targeted micro-balloons and micro-capsules” and non-invasive ultrasound diagnostics to diseases of the cardiovascular system include targeted therapy of atherosclerotic plaque, monitoring thrombosis and haemorrhagic events, diagnosis and treatment of stroke, delivery of anticoagulants, and clot destruction. The medical applications of such devices are countless and many other are close to come. Societal advantages are closely related: lowering the costs of marketed ultrasound contrast agents (today on sell at about 135 Euros/kit) due to longer shelf life and circulation life, easiness of the device preparation, localized delivery of drugs with increased bioavailability, patient acceptability (this issue is particularly important in young patients and children), and consequently healthcare costs reduction.
Arrays based devices of self-assembled micro-balloons and microcapsules for ex situ testing and monitoring is also part of this new concept applied to theranostics. Both devices impact on the “conventional” diagnosis imaging by probing the molecular specific cellular and tissue malfunctions at the very early stages of the disease, rather than imaging the end effect of these molecular alterations

Proof of concept.

The final prove and assessment of the S.I.G.H.T. activities based on the concept described above will be:

• a prototype of the new multifunctional devices (micro-balloons and micro-capsules) emerging from the project studies.
• Knowledge for the validation of product/procedure in view of an industrialization plan.
• Knowledge for a next-step pilot production.
• Contributions to the formulation of appropriate standards to the European Committee for Medical Ultrasound Safety (ECMUS), in view of an ad hoc formulation of guidelines to be assessed for a “theranostic” approach.

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