WO2017178954A1

WO2017178954A1 - Nanobubbles and uses thereof

Info

Publication number: WO2017178954A1
Application number: PCT/IB2017/052060
Authority: WO
Inventors: Maria Carafa; Andrea BETTUCCI; Carlotta Marianecci; Federica Rinaldi; Angelo BIAGIONI
Original assignee: Università Degli Studi Di Roma "La Sapienza"
Priority date: 2016-04-11
Filing date: 2017-04-10
Publication date: 2017-10-19
Also published as: ITUA20162486A1

Abstract

Nanobubbles characterised by a double lipophilic layer surrounding a central gaseous portion, with an aqueous layer placed between the said central portion and the said lipophihc double layer, are the subject of the invention. The lipophilic double layer may contain within it compounds of a lipophilic nature. The aqueous layer located between the lipophilic double layer and the central gaseous portion may contain compounds of a hydrophilic nature within it. The nanobubbles may also further comprise targeting molecules associated with the lipophilic double layer, preferably polypeptides, polynucleotides, antibodies or antibody fragments.

Description

NANOBUBBLES AND USES THEREOF

^• i i i i

Technical field of the invention

The invention relates to nanobubbles and uses thereof; the invention also relates to the process of producing said nanobubbles.

In particular the invention relates to stabilised nan op article systems of a polymer nature capable of acting as vehicles for agents suitable for use in the diagnostic, therapeutic and theranostic fields.

Known art

Sonography is an imaging technique that has an essential role in the diagnosis of many types of diseases and is a clinical research method based on the detection of ultrasound waves reflected by the various tissues making up a biological system. In general, in all ultrasound techniques, reconstruction of the images of various parts of the body is based on the detection of ultrasound waves reflected at tissue discontinuities present within them. This technology is noninvasive and convenient, but has some limitations - for example it is impossible to reach and therefore display blood vessels having a diameter of less than 200-300 microns.

The possibility of obtaining good quality ultrasound images, even for those parts of the human body which because of their anatomy and physiology are otherwise difficult to study, is thus not only the result of the high technological level achieved in the construction of present-day ultrasound machines, but also the development of a class of agents capable of improving the quality, that is the contrast, of ultrasound images, with a consequent increase in the diagnostic power of this investigation technique; these agents are known as Ultrasound Contrast Agents (UCAs).

These agents, which comprise gas microbubbles coated with a "shell" of variable composition, injected intravenously, make it possible to improve the qualitative display of various parts of the body in direct contact with tissues having similar acoustic characteristics and zones characterised by microvascularisation, which is a characteristic of some organs such as for example the heart, or some diseases, for example prostate diseases.

The dispersion of gas-containing microbubbles in blood gives rise to a specific acoustic impedance discontinuity between the bubbles and the blood, this acoustic impedance being the physical quantity parameterising the resistance offered by a medium to the propagation of a wave. This is defined as the ratio between the acoustic pressure at a point and the consequent velocity of movement - around the equilibrium position - of the particles of the medium in which the wave is being propagated; this quantity is equal to the product of the density of the medium in which the elastic wave propagates and the wave propagation velocity; it is measured in rayls (Ry): 1 Ry = 1 kg/(m² s).

The specific acoustic impedance discontinuity between gas microbubbles and blood, which can exceed values of 10³ Ry, causes great diffusion of the elastic energy generated by the ultrasound probe that is incident on the microbubble-blood system, and there is therefore an increase in the intensity of the reflected echo; this in turn is translated into an increase in the contrast of the ultrasound image of the blood vessel affected by the elastic wave in comparison with that found when bubbles are absent.

There are different types of bubbles currently in use in diagnosis; they are however rather unstable and only allow an improvement in the ultrasound image to be obtained for a short period of time.

A further limitation is associated with the micrometre size of such agents, which are unable to improve the ultrasound image of vessels having dimensions smaller than a few microns.

The problem associated with microembolisms or risks associated with administering particles having dimensions similar to those of capillaries (1-10 μιη) into the circulating flow, which might cause occlusions within them, should also not be underestimated.

Commercially available preparations comprise microbubbles covered with a single layer of molecules of an amphiphilic nature, purely for the purpose of rendering the contrast agent more stable (1 - 6).

These structures however offer little stability in terms of gas retention and must therefore be reconstituted at the time of use (7 - 8).

Publications relating to the production of bubbles nanometre- sized bubbles (or nanobubbles) from liposomes that are capable of acting as a vehicle for a gas relate to structures of dimensions varying between 400 and 800 nm, produced using very complex formulations (9 - 11).

Work by Schroedera et al. in 2009 proposed the combination of liposomes and ultrasound, but the liposomes are suggested as an external stimulus to modulate the release of medicinal compounds (12).

A recent patent application relating to nanometre-sized bubbles relates to structures coated with a single layer of molecules of an amphiphilic nature (13).

The nanobubbles described therein comprise a single layer and therefore demonstrate little or inadequate stability. They are also characterised by a compartment of an apolar nature of insufficient size to include any lipophilic medicinal compounds. The bubbles prepared in this way in the aforementioned application also show too wide a size distribution and therefore reduced applicability in vivo because it is difficult for them to reach the microcirculation, they may give rise to the risk of causing microembolisms, and also they do not show that water is present in the internal compartment and they are therefore unable to act as a vehicle for polar medicinal compounds.

This invention is therefore provided to resolve the problems in the known art.

Summary of the invention

The invention relates to nanobubbles characterised by a double lipophilic layer containing an aqueous layer and a central gaseous portion as illustrated in Figure 1.

Another object of the invention is compositions and formulations containing the abovementioned nanobubbles.

Another object of the invention is the process for producing nanobubbles. Yet another object of the invention is the use of nanobubbles in the diagnostic, therapeutic and theranostic fields.

Other objects will be obvious from the following detailed description.

Brief description of the figures

Figure 1 - Diagrammatical illustration of the structure of the nanobubbles in which an internal gaseous compartment surrounded by a layer of water (suitable for acting as a vehicle for medicinal compounds or a diagnostic agent of a hydrophilic nature), both enclosed by a double lipophilic layer (capable of acting as a vehicle for a medicinal compounds or diagnostic agent of a lipophilic nature).

Figure 2 - Experiment on the stability of nanobubbles analysed by SAXS (Small Angle X-ray Scattering) after thirty days' storage at a temperature of 4°C; Graph A: Span 20 nanobubbles. Graph B: DMPC nanobubbles.

"q" is the transferred vector momentum as a function of the intensity of the beam; the intensity of "q" on a logarithmic scale is proportional to q-ⁿ where "n" is the gradient of the straight line providing information on the nature of the object being examined.

Figure 3 - Experiment on the stability of Span 20 nanobubbles examined by pulse-echo after ninety days' storage at a temperature of 4°C; the nanobubbles retain the same acoustic efficiency if examined after a period of approximately three months following their preparation.

Figure 4 - Experiment on the stability of DMPC nanobubbles carried out over a period of three months at different storage temperatures, by DLS (dynamic light scattering) analysis.

Figure 5 - Frequency response of the photoacoustic cell for a disperse suspension of SonoVue^® over time, compared with the case where the cell is filled with water alone. Two resonance peaks for the photoacoustic cell are present (inherent vibration modes) in the band between 200-300 Hz and 400-600 Hz.

Figure 6 - Stability experiment on SonoVue^® microbubbles analysed by pulse-echo at a temperature of 37°C; the medicinal product rapidly loses acoustic efficiency over time. Figure 7 - Frequency response of the photoacoustic cell corresponding to two different solutions present within it: Graph A, dispersed suspension of SonoVue^®; Graph B, dispersed solution of nanobubbles. Two resonance peaks of the photoacoustic cell (inherent vibration modes) are present in the band between 200-300 Hz and 400- 600 Hz.

Figure 8 - Attenuation of ultrasound intensity at 14 MHz as a function of temperature for a sample of nanobubbles having a Hepes concentration of 37%.

Figure 9 - Attenuation (expressed in dB/cm) measured at the frequency of 14 MHz for Span 20 nanobubbles at various concentrations in Hepes buffer solution.

Figure 10 - Attenuation frequency spectrum of the ultrasound signal of Span 20 nanobubbles measured using the pulse technique.

Figure 11 - Diagram comparing the acoustic efficiency of a sample of Span 20 nanobubbles with only gas or with probes: at the top SPAN 20 + CHOL + PFC; in the middle SPAN 20 + CHOL + PFC + NILE RED; at the bottom

SPAN 20 + CHOL + PFC + calcein.

Figure 12 - Experiment on stability of DMPC nanobubbles performed over a period of three hours, 180 minutes, at a temperature of 37°C in the presence of 45% (v/v) human and bovine serum, using DLS (dynamic light scattering) analysis.

Figure 13: Transport of calcein through the BBB after administration to mice, using lipid and surfactant nanobubbles. A) Fluorescence microscope images showing the location of the calcein in thin coronal sections of the brain. B) Quantification of calcein in the brain in terms of detected fluorescence.

Detailed description of the invention

The invention relates to nanobubbles characterised by a double lipophilic layer surrounding a central gaseous portion, with a layer of water being located between the said central portion and the said lipophilic double layer (Figure 1). The lipophilic double layer may contain compounds of a lipophilic nature within it. The aqueous layer located between the lipophilic double layer and the central gaseous portion may contain compounds of a hydrophilic nature within it.

The nanobubbles may also further contain targeting molecules located on the surface of the lipophilic double layer, preferably polypeptides, polynucleotides, antibodies or antibody fragments, which can be prepared using techniques known to those skilled in the art.

The invention is based on having selected components which make it possible to obtain nanobubbles with a lipophilic double layer using the process according to the invention; these components stabilise the structure of the nanobubbles making it possible to prevent escape of the gas contained within them, thus imparting stability over very long periods. This is confirmed by the stability tests illustrated in Figures 2A, 2B, 3, 4 and 10.

Using the process according to the invention and with the said components it is not only possible to prepare nanobubbles of nanometre size but also to obtain maximum dimensional uniformity in the preparation, as indicated by the polydispersity index shown in Table 2, which is always below 0.25, and preferably 0.2 or less.

The components are selected from the following phospholipids: 1 , 2 -dimyristoyl-sn.- glycero- 3 -phosphocholine (DMPC) , dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dicetyl phosphate (DCP), in combination with cholesterol.

Nanobubbles produced using any one selected from the following surfactants - Span 20 and 60 and Tween 85, 81, 80, 65, 61, 60, 21, 20 - are similarly stable.

For the purposes of this description by Tween 85 is meant polyoxyethylene glycol sorbitan trioleate; by Tween 81 is meant polyoxyethylene (polyoxyethylene units = 4) glycol sorbitan monooleate; by Tween 80 is meant polyoxyethylene (polyoxyethylene units = 20) glycol sorbitan monooleate; by Tween 65 is meant polyoxyethylene glycol sorbitan tristearate; by Tween 61 is meant: polyoxyethylene(polyoxyethylene units = 4) glycol sorbitan monostearate; by Tween 60 is means polyoxyethylene (polyoxyethylene units = 20) glycol sorbitan monostearate; by Tween 21 is meant polyoxyethylene (polyoxyethylene units = 4) glycol sorbitan monolaurate; by Tween 20 is meant polyoxyethylene (polyoxyethylene units = 20) glycol sorbitan monolaurate.

For the purposes of this invention by Span 20 is meant [2- [(2R,3R,4S)-3,4-dihydroxyoxolane-2-yl]-2-hydroxyethyl] dodecanoate; by Span 60 is meant sorbitan monostearate.

The gas inserted within the nanobubbles belongs to the class of perfluorocarbons, preferably tetradecafluorohexane is used.

The method for preparing the nanobubbles according to the invention comprises the following essential steps:

(a) Preparing a film by dissolving an aliquot of cholesterol and one or more cationic phospholipid surfactants in an organic solvent and subsequent evaporation, this preparation step being characteristic of preparation using film and being known to those skilled in the art and described in (15);

(b) Hydrating the film obtained in stage (a) with water, the water may be replaced by an aqueous solution obtaining a water-soluble therapeutic and/or diagnostic agent such as a diagnostic probe (for example a fluorescent, radioactive, or luminescent probe or other known therapeutic or diagnostic agents);

(c) Placing the mixture obtained in stage (b) in a sealed container or reactor, then adding an aliquot of a straight, branched or cyclic C3-C7, preferably Ce chain perfluorocarbon PFC, liquid in equilibrium with its gas phase, at atmospheric pressure;

(d) Subjecting the suspension obtained in the previous stage to sonication at ambient temperature, for example using an ultrasound sonicator provided with a microprobe and operating at a frequency of 20 kHz and an amplitude of 16% (Vibracell-VCX 400-Sonics, USA) to obtain unilamellar structures; this stage may possibly be replaced by processes having the same effects, for example extrusion with repeated filtration passes through a membrane with a cut-off from 1.5 μιη to 0.1 μιη, or any other technique known to those skilled in the art;

(e) The suspension obtained in the previous stage is then cooled, avoiding freezing, preferably to a temperature of not less than 4°C.

The suspension obtained in paragraph (e) can undergo a further purification step to select a population of nanobubbles of uniform dimensions; for example nanobubbles can be selected by centrifuging the suspension, preferably at 600 rpm for 20 minutes at 25°C and then removing the supernatant; alternatively, if the process in paragraph (d) provides for extrusion, the centrifuging stage may be omitted.

Where not otherwise specified the steps in the process are preferably carried out at ambient temperature.

The nanobubbles so obtained are characterised by measurements of dimensions and ζ potential, SAXS and acoustic measurements.

Therapeutic and/or diagnostic agents of a lipophilic nature are added by adding suitable aliquots in stage (a).

Using the procedure described the inventors have surprisingly found that the nanobubbles prepared are characterised by the fact that they have a central core holding gas surrounded by an aqueous layer, all enclosed by a double layer (as discovered experimentally by Small Angle X-Ray Scattering, SAXS, investigations, Figures 2A and 2B).

Figure 2 (Graphs A and B) shows spectra having characteristic peaks corresponding to an internal distance within the particles, which provides indications about their local structure. A change in intensity such as q-⁴ indicates the presence of a sharp interface between two media. It is therefore reasonably possible to state that the particle comprises a double layer in the presence of a sharp interface between two media (double layer/gas). The formation of nanobubbles containing gas, which are still stable after 30 days, in that the local structure is the same, is therefore proven.

The nanobubbles according to the invention are capable of overcoming the limitation of existing contrast media in that they are more stable over time and extremely uniform in shape and dimensions in comparison with those in the known art; in particular these characteristics allow them also to be used when applied to paediatric and veterinary diagnostics, in particular small animals, avoiding the risk of the formation of microembolisms which might occur following the use of preparations of micrometre-sized or nanobubble preparations which do not have uniform dimensions.

The possibility of adding both water-soluble and liposoluble therapeutic and/or diagnostic agents to the nanobubbles according to the invention makes it possible to produce vectors for use in diagnostic, therapeutic and theranostic applications.

In therapy, in particular, nanobubbles can behave like cavitation nuclei capable of being activated by exposure to low frequency power ultrasound (US). The cavitation induced by the US which results in the destruction of the gas bubbles is capable of opening cell membrane pores of approximately 300 nm in diameter.

This phenomenon, known as "sonoporation", may be functional in increasing cell uptake or the passage of medicinal compounds and genetic material across biological membranes; in some cases it is very difficult to overcome the aforesaid membranes; for example the blood- brain barrier (BBB) is a barrier which is difficult to overcome using strategies currently available. We have surprisingly found that using the nanobubbles according to the invention sonoporation of the BBB is achieved in an effective, transient and reversible manner.

The composition of the proposed systems has the advantage that it can be easily apphed industrially, making use of phospholipids or surfactants added to variable fractions of cholesterol.

As shown in Figure 1, the nanometre-sized nanobubbles according to the invention are characterised by an apolar double layer or bilayer membrane comprising phospholipids and/or surfactants in combination with cholesterol.

The inventors have carried out different tests in order to select the type and quantity of phospholipid or surfactant capable of providing a double layer in the presence of cholesterol making it possible to obtain the structure shown in Figure 1 and the corresponding innovative characteristics illustrated below.

Numerous components mixed together in the presence of cholesterol in various molar ratios were tested in order to obtain the nanobubbles. The following phospholipids and surfactants were tested: l,2-dipalmitoyl-S7 -glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl- S7 -glycero-3-phosphoethanolamine (DPPE), l,2-dimyristoyl-sn.-glycero- 3-phosphocholine (DMPC), dicetyl phosphate (DCP), 1,2-dipalmitoyl- sn.-glycero-3-phosphate (DPPA), phosphatidylcholine (PC), 1,2- distearoyl-S7 -glycero-3-phospho-(l'-rac-glycerol) (PG), 1,2- dioctadecanoyl-S7 -glycero-3-phospho-(l'-rac-glycerol) (DSPG), 1,2- dioleoyl-S7 -glycero-3-phosphocholine (DOPC), 1,2-dihexanoyl-sn.- glycero-3-phosphocholine (DHPC), l,2-dilauroyl-sn.-glycero-3- phosphocholine (DLPC), l,2-dioctadecanoyl-sn.-glycero-3-phosphate (DSPA), l,2-distearoyl-S7 -glycero-3-phosphoethanolamine (DSPE), 1,2- diarachidoyl-S7 -glycero-3-phosphocholine (DAPC), 1,2-c palmitoyl-sn.- glycero-3-phosphate (DPPA), polyoxyethylene glycol sorbitan monopalmitate (Tween 40), polyoxyethylene cetylether (Brij 35), polyoxyethylene monostearate (MYKJ 52), sorbitan monolaurate (Span 20), sorbitan monooleate (Span 80), sorbitan trioleate (Span 85).

The combinations yielding the best results are reported in Table

I.

From the experiments performed it emerged that of the components tested the combinations of DMPC+cholesterol, DMPC+DCP+cholesterol, DPPC+cholesterol and Span20+cholesterol in particular are capable of giving rise to the nanobubbles according to this invention when present in particular molar ratios (Table 1).

Table 1

Nanobubble structure and dimensions

The dimensions of the nanobubbles according to Dynamic Light Scattering (DLS) measurements are approximately 150 nm (130-260 nm) and the surface charge expressed as the ζ potential value is from weakly to markedly negative (-4 / -71 mV). (Table 2). Table 2.

The polydispersity index (pdi) associated with the measurements of dimensions is always below 0.25, preferably 0.2 or less. This index is important in that it provides an idea of the range of dimensions in the nanobubble population. In particular it is important to have a vesicular suspension with a monodisperse population so that it will behave uniformly both during storage and after administration in vivo in the distribution and cell intern alisation stages. The vesicular structure so produced is characterised by a double layer of a lipophilic nature including an internal compartment in which an aqueous layer and the gas coexist (Figure 1), these structural characteristics being confirmed, as previously mentioned, by studies carried out using Small Angle X- ray Scattering (SAXS) (Figure 2).

Stability

Stability tests surprisingly proved that the structures produced are extremely stable.

As established by the DLS analysis, Figure 4, pulse echo analysis, Figure 3, and SAXS analysis, Figure 2, the stability of the nanobubbles in suspension during storage is in fact extremely high, at least 3 months at a storage temperature of 4°C. Figure 2 shows that the SAXS spectra for nanobubbles stored for 1 month are the same as those prepared 1 day before. Figure 3 clearly shows that the nanobubbles maintain the same attenuation after 90 days.

Figure 4 shows that dimensions and zeta potential do not significantly change after 90 days' storage.

They are also stable for at least 180 minutes at temperatures simulating in vivo conditions (37°C and 40°C) (Figure 8) and for 180 min in the presence of 45% (v/v) (fig.12) human and bovine serum. These are very much better data than those reported in US2014/0147390 and WO2014/054026.

Acoustic efficiency and interaction with ultrasound

The system has been tested in terms of acoustic efficiency and it has surprisingly been found that the nanobubbles are stable, in terms of maintaining dimensions and zeta potential, persistence of acoustic signal, and gas retention that is very much better than that of known products. The experiment with ultrasound illustrated in Figure 3, using the pulse echo technique, points out that the attenuation measured at 14 MHz does not undergo any changes after 90 days for any of the concentrations tested, and in comparison with Figure 6 for SonoVue^®, diluted according to the manufacturer's specifications and measured using the same technique, it is found that the latter's stability is reduced after approximately 100 hours. Figure 5 shows that using a technique of the photoacoustic type the stability of SonoVue^® is reduced after 100 hours, confirming what was obtained using the previous technique.

Efficiency of the nanobubbles from the point of view of interaction with ultrasound was evaluated using an investigation technique widely used to characterise the products currently on the market in the same class as those described in this description. This is based on the transmission and reception of short ultrasound pulses caused to propagate through a solution of nanobubbles (this technique is also known as the pass-echo technique) (16).

Analyses of the differences in information content between the emitted signal and the received signal provide the acoustic characterisation of the nanobubbles: the parameters describing the efficiency of nanobubbles are the attenuation experienced by the acoustic signal passing through the therapeutic and/or diagnostic agent (greater attenuation is reflected in greater ultrasound image contrast), and the corresponding frequency band at which this is obtained.

The efficiency of trapping gas within the nanobubbles was instead evaluated using a photoacoustic technique (17): this choice was due to the fact that the photoacoustic signal generated by a liquid periodically heated by a coherent (laser) light is extremely sensitive to the quantity of gas present in a liquid solution. The results found were compared with those for a clinically approved ultrasound contrast agent comprising a liquid suspension of micrometre-sized bubbles of SF6 gas coated with a single layer of phospholipids (SonoVue^® - Bracco Imaging, Milan, Italy) (Figure 7 Graphs A and B). It will be seen from the figure that the two resonance peaks for the photoacoustic cell (around approximately 300 and 600 Hz) shown by the cell when filled with only distilled water (Figure 7 Graph A, open circles) or with a solution of Hepes ([N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid)], 10 mM, pH 7.4) (Figure 7 Graph B, open circles), experience attenuation (indicated by ΔΑι and ΔΑ2 in both Figure 7A and Figure 7B) when the cell is filled with SonoVue® (Figure 7 Graph A, solid circles) or a solution of nanobubbles (Figure 7 Graph B, solid circles).

Because the attenuation of the resonance peaks is proportional to the quantity of gas present in the liquid (17), both that provided by the SonoVue® microbubbles (Figure 7 Graph A, solid circles) or the nanobubbles according to the invention (Figure 7 Graph B, solid circles), comparison shows that there is a greater capacity for trapping gas within the shells of the nanobubbles according to the invention in comparison with SonoVue®.

The measurements made have provided precise indications regarding both the objective acoustic properties of the vesicular nanobubbles and the improvements which this new formulation introduces into the scope of medical diagnosis through imaging. In the latest generation ultrasound instruments the operating frequency band in fact has an upper limit of a frequency of 20-25 MHz, well above the frequency bands of existing ultrasound contrast agents within the range 2-6 MHz corresponding to diameters of the order of 3-6 μιη (SonoVue^®). From the measurements performed the vesicular nanobubbles according to the invention instead have dimensions at least twenty times smaller (diameters of approximately 100 nm), provided an operating frequency band in the range 15-30 MHz; this frequency is not only more compatible with present day ultrasound machines, but is also capable of ensuring operation at maximum efficiency with ultrasound machines which will achieve even higher frequency bands in the future, up to 40 MHz.

The nanobubbles according to the invention guarantee operation between 5 and 40 MHz, better if at 20 MHz. These small dimensions make it possible to use nanobubbles diagnostically in paediatric and veterinary contexts, where the dimensions of the peripheral blood vessels are small (20).

In addition to the frequency band characteristics, the results obtained show a considerable increase in the diagnostic power of the nanobubbles according to the invention in comparison with that demonstrated by micrometre-sized contrast agents. In fact measurement of attenuation of the ultrasound signal passing through the DMPC nanobubbles sample provides values of around 18 dB/cm (attenuation of the acoustic signal for every centimetre thickness of the layer of sample passed through), which, for the same quantity of micro- and nanobubbles in suspension should be compared with the value of approximately 5-6 dB/cm provided by SonoVue^®, measured using the same experimental technique. Nanobubbles comprising DMPC or DPPC or Span 20 on the other hand provide attenuation values of 18 dB/cm for those phospholipids and 6-8 dB/cm for surfactants.

Finally, when the acoustic efficiency of the nanobubbles according to the invention were measured as a function of time it was found that they are capable of maintaining their acoustic properties unchanged for times which are wholly compatible with their diagnostic and therapeutic applications, demonstrating that they remain efficient for times of several hours.

Figure 10 shows that the nanobubbles, in this example of Span 20, have unchanged attenuation when the frequency of the ultrasound is varied and the dilution is increased.

Acting as a vehicle for medicinal compounds and/or probes

Thanks to their structure (Figure 1) the nanobubbles according to this invention are capable of acting as vehicles for both medicinal compounds of a hydrophilic nature in the aqueous layer within the bilayer, and lipophilic medicinal compounds in the double layer, at the same time as a gaseous compound, and are therefore capable of being used in the therapeutic, diagnostic and theranostic fields. The nanobubbles may for example contain perfluorocarbons and medicinal compounds of a lipophilic and hydrophilic nature at the same time.

Molecules of the fluorescent type of a hydrophilic and lipophilic nature have also been included in the proposed structures, in addition to the gas, to assess whether physical chemical properties and/or stability remained unchanged.

In order to demonstrate this capability, model hydrophilic and lipophilic molecules, calcein and Nile red, were included within the nanobubbles together with the gas. The nanobubbles charged with the fluorescent probes were characterised in terms of acoustic efficiency, comparing it with that of the nanobubbles as such (Figure 11).

The experiment shown in Figure 11 has shown that the joint transport of probes does not have an adverse effect on the acoustic efficiency of the nanobubbles and does not interfere with the structure of the gaseous compartment in that the probes, depending upon their characteristics, are located selectively in the aqueous or lipophilic compartment.

In addition to this, the materials used for preparation of the nanobubbles and the gas are not toxic.

It can be concluded that the nanobubbles proposed have the following advantages:

(a) They have nanometre-sized dimensions and comprise a double layer of a lipophilic nature made up of amphiphilic molecules;

(b) The formulations proposed, prepared using the process according to the invention, are capable of providing nanobubbles with a stable double layer, in comparison with those known to the inventors. They are in fact highly stable in aqueous solution at a storage temperature of 4°C (at least 3 months) and at temperatures simulating in vivo conditions (37 and 40°C) up to 180 min (Figure 8); and also in the presence of human and bovine serum for 180 minutes at a temperature of 37°C (Figure 12). The nanobubbles according to the invention are stable without the need to make use of preservative and reconstitution techniques such as lyophilisation and subsequent dehydration and the addition of gas. (c) In comparison with known formulations and in particular those described in patent applications US2014/0147390 and WO2014/054026, the nanobubbles according to the invention are produced with highly uniform dimensions (Table 2), a property which allows them to be used in the veterinary and paediatric fields, avoiding the risk of microembolisms.

(d) They show no changes in physical-chemical properties and stability after the inclusion of model molecules of a hydrophilic and lipophilic nature;

(e) Their dimensions do not change during photoacoustic experiments;

(f) Use of the nanobubbles according to the invention makes it possible to achieve sonoporation of the blood-brain barrier in an effective, transient and reversible manner;

(g) They are capable of trapping a larger quantity of gas, for example more than 30% in comparison with SonoVue^®, despite their smaller dimensions (Figure 7).

The latter point was assessed using a photoacoustic technique measuring the decreasing amplitude of the resonance peaks corresponding to the intrinsic frequencies of an acoustic resonator used as a photoacoustic cell as the concentration of nanobubbles in the cell was varied. The photoacoustic cell, of brass, was an acoustically resonant structure of cylindrical shape having an internal volume of V = 7.68 cm³, within which was located the liquid solution which has to be characterised photoacoustically; an argon ion laser (wavelength

4880 A units), periodically modulated in amplitude at a variable frequency between 50 and 2000 Hz, struck the liquid through a transparent window of the cell. The periodic heating of the liquid caused periodic variations in its volume which were reflected in displacements of a small polyvinyl membrane in contact with the liquid; these displacements were measured with a laser vibration meter. The measurement relating to gas content within the nanobubbles was based on the fact that the more gas is present in the micro/nanobubbles dispersed in the liquid, the greater will be the decrease in amplitude of the oscillation of the polyvinyl membrane at the resonant frequencies of the photoacoustic cell (17- 19).

In the measurements made, 1.4 ml of SonoVue^® solution were diluted in 7.68 ml of distilled water causing relative variations in vibration amplitude for the first and second resonance frequencies of the photoacoustic cell equal to (Λ^ « 0.77 and (AA₂ / A₂ )_SV « 0.48 respectively (Figure 7a). As far as the nanobubbles are concerned, 0.7 ml of nanobubbles solution in 7.68 ml of Hepes generated a relative variation in vibration amplitudes at the two resonance frequencies of

« 0.54 and (AA₂ / A₂ )_M « 0.33 respectively, (Figure 7b).

Because the measured values for the nanobubbles were approximately 30% lower than those obtained with SonoVue^®, the greater capacity for trapping gas within the nanobubbles must therefore be estimated to be 30% greater than that of SonoVue^® for the same volume of micro/nanobubbles dispersed in the liquid.

The following examples will be provided as an illustration of the invention and are not to be regarded as limiting its scope.

Examples

Sample preparation

The nanobubbles in Table 1 (DMPC, DPPC, Span and Choi) were prepared using the "film" technique as described in the literature (14, 15). In particular the components of a lipophilic nature, that is phospholipids or surfactants and cholesterol in the ratios described in Table 1, and therapeutic and/or diagnostic agents of a lipophilic nature were dissolved in a mixture of organic solvents (3/1 v/v chloroform/methanol), subsequently evaporated in a rotavapor to give rise to the formation of a uniform thin layer of the components, called a film, on the walls of the preparation container. The film obtained was hydrated by adding an aqueous phase (10 mM Hepes buffer pH 7.4, or aqueous solutions of therapeutic and/or diagnostic agents of a hydrophilic nature) when required in order to obtain the nanobubbles.

Liquid PHF (tetradecafluorohexane) in equilibrium with its gas was added to the film to obtain different nanobubble formulations. In one particular mode this technique provides for the insertion of liquid and gaseous PHF at the same time through drawing PHF in the liquid phase into a syringe, where a saturated environment is created within it with an equilibrium between liquid and gaseous PHF. The PHF present in the body of the syringe (gas and a suitable quantity of liquid PHF) was introduced into the suspension vesicles. The dispersion was then stirred mechanically and sonicated for 15 minutes at a temperature of 25°C using an ultrasound sonicator provided with a microprobe operating at a frequency of 20 kHz and an amplitude of 16% (Vibracell-VCX 400-Sonics, USA). The nanobubbles obtained were then subjected to cooling by means of a thermal shock in a bath with water and ice, for a period of 2 minutes. The nanobubbles were then purified from the excess material by centrifuging using an MPW-260R centrifuge (MPW Med. Instruments) at 600 rpm for 20 minutes at 25°C.

Experiment to establish dimensions and structure

Dimensioning using DLS and structural analysis using SAXS.

Dynamic light scattering is used to study the structure of colloidal suspensions and more generally aggregates having dimensions which can vary from a few nm to a few micrometres.

From a study of the fluctuations in such a signal information is obtained on the shape, dimensions and state of aggregation of the particles included in the sample.

DLS measurements were carried out on samples having the compositions shown in Table 1 prepared according to the method previously described and then purified. These are measurements which were performed on both empty samples and those charged with fluorescent agents and gas. The analyses were performed at 25°C; each analysis relates to at least 3 measurements from which the software derives a mean value representative of the diameter of the vesicles.

The distribution curve for nanobubble dimensions is characterised by a polydispersity index (PDI), which should not be greater than 0.25 for monodispersed populations.

These measurements have been useful for initial characterisation of the vesicles prepared, and to carry out stability studies over time and at different temperatures. The change in the dimensions of the sample, which will be smaller the more stable the sample, in that it will not experience changes in dimensions over time caused by phenomena of a degradative type, is detected in these studies.

DETERMINATION OF ζ POTENTIAL

The presence of charged particles in a suspension is of great importance for the stability of the dispersion itself, as the particles will tend to repel each other.

An important consequence of the presence of electrical charges on the surface of the particles is that they will demonstrate effects, known as "kinetic effects", under the influence of an applied electric field.

Measurement of the ζ potential provides information on the physical-chemical characteristics of the particles in suspension and is extensively used to predict the stability of systems in relation to aggregation phenomena.

The stability of suspensions is strongly influenced by the surface charge; the greater the charge present on the surface of the vesicles the greater will be their tendency to remain separated because of the repulsion effect occurring between particles having the same charge.

If all the particles in suspension have very high negative or positive ζ potentials they will tend to repel each other and there will be no coalescence or flocculation.

A suspension is generally considered stable when the ζ potential values are less than -30 mV or greater than +30 mV.

MEASUREMENT EQUIPMENT

Measurements were performed using the same computerised inspection system (Malvern Zetasizer Nano ZS 90, Malvern, UK) used for DLS.

ζ potential measurements were made at the temperature of 25°C on samples stored at 25°C and 4°C, or 37°C in the case of the stability studies at body temperature, after dimensioning using the DLS technique. These measurements were performed during the characterisation and stability studies.

Because each element has its own internal structure, which can be determined by making use of radiation of a wavelength comparable to the measurements of interest, Small Angle X Ray Scattering (SAXS) experiments, a technique which can be used to gain access to a distance range of the order of nm, were carried out in order to investigate the structural and morphological characteristics of the samples containing gases.

The ERSF (European Synchrotron Radiation Facility, Grenoble, France) ID02 facility was used for the experiments in this work.

The ESRF synchrotron is capable of producing extremely bright X-rays, that is a thin and very intense beam.

In the study the SAXS measurements were obtained by keeping the position of the sample fixed and automatically moving the detector in a vacuum tube from 0.75 m and 10 m from the sample, so as to be able to gain access to a wide range of scattering wave vectors: 0.1 nm- ^l< q < 3 nm-¹, where q = (4π/ λ) sin(0 12), with λ being wavelength (~0.1 nm) and Θ the scattering angle. The samples were placed in plastics capillaries (KI sheet, ENKI srl, Concesio, Italy) of internal diameter of 2 mm, thickness 0.05 mm and with 98% X-ray transmission, sealed with polyethylene caps.

The capillaries were then mounted horizontally in a 6-station sample holder and were filled after they had been installed. In this configuration it is possible to guarantee almost simultaneous measurements of empty and solvent samples under the same conditions; this is very important in order to be able to subtract backgrounds in an optimum way and compare the results obtained.

In order to be able to carry out precise temperature measurements an ad hoc built thermostat suitable for fitting to the ID02 line was used; the temperature stabilisation error within the capillaries was less than 0.05°C.

The measurements were made at 25°C with a very short exposure time, 0.1 s, to minimise any damage due to the use of radiation.

The SAXS profiles obtained show the diffracted radiation intensity as a function of momentum transfer, q. Some spectra for empty and solvent cells were also obtained, carefully compared and subtracted from the spectra of each sample. The analyses performed made it possible to obtain information on the dimensions, uniformity and shape of the gas-containing samples in suspension.

Results of stability analyses

Stability analyses in terms of changes in dimensions over time at two different storage temperatures were carried out using DLS (Figure 4), while confirmation that the initial gas concentration within the nanobubbles was maintained after storage for three months was obtained from acoustic attenuation experiments (Figure 3). The vesicular of samples prepared, both empty and containing gas, were subjected to dimensioning and ζ potential analyses after purification.

Study of the dimensions and surface charge of the vesicular systems was extended over time and the abovementioned parameters were assessed over a time interval of approximately 3 months at two different temperatures in order to study the stability of the samples.

This study was performed holding identical aliquots of the samples in test tubes held at ambient temperature on a laboratory bench and in a refrigerator at 4°C.

Initially dimensioning and ζ potential measurements were made every day for the first week of storage and then at a frequency of once per week.

Through this study the stability of the samples in terms of any changes in the parameters which we were investigating probably caused by the occurrence of degradation phenomena, or aggregation and coalescence, was followed over time.

Changes in dimensions and zeta potential were also studied to assess the stability of the nanobubbles in the presence of both human and bovine sera at a concentration of 45% (v/v) over a time period of 3 hours at a temperature of 37°C (Figure 12).

This study enabled us to follow the stability of the samples in terms of any changes over time in the parameters which were investigating, probably as a result of the occurrence of adsorption and degradation phenomena following interaction between the nanobubbles and plasma proteins. From the data shown in Figure 12 it is clearly apparent that the nanobubbles are stable if placed in contact with human and bovine serum over the time interval investigated.

Acoustic characterisation results and interaction with ultrasound

Because nanobubbles full of gas dispersed in a liquid act as diffusion (scattering) centres for an elastic wave propagating in the liquid and incident upon them, the efficiency of gas trapping within the nanobubbles was also evaluated using a previously described high frequency ultrasound pulse technique (7). This technique, which makes use of the same physical principle (measurement of the intensity of the reflected echo) as is also used for the formation of ultrasound images can be used to evaluate the efficiency of the nanobubbles as a contrast medium in medical ultrasound imaging; in fact the increased attenuation of the ultrasound signal transmitted by the solution containing nanobubbles is associated with the increased efficiency with which the incident elastic energy is scattered into space, consequently increasing the ultrasound reflection capacity (increased echogenicity) to the source of the waves themselves, which in the clinical situation will be the sensor connected to the ultrasound unit used for medical investigation. The two-way correspondence between the increased attenuation of the acoustic signal measured by the pulse-echo technique and the increase in the scattering capacity of the nanobubbles also makes it possible to use the ultrasound pulse technique to measure the frequency spectrum of the nanobubbles' scattering efficiency, or to determine the ultrasound frequency at which the elastic energy is most diffused by the nanobubbles into space.

An ultrasound transducer (PanametricsU8423040 V319) having a central frequency of 14 MHz and a pass band of between 11 and 17 MHz at -6 dB was used for the measurements. The transducer, which was fed from an ultrasound pulse generator/receiver (Panametrics500 PR), acts as both an emitter and receiver of elastic waves. During the emission stage this generates short ultrasound pulses (lasting a few microseconds) which propagate in the dispersed liquid solution of nanobubbles present in a small hollow cell - of cylindrical shape - made in a Plexiglas tube. One of the base surfaces of the cell comprises the transducer surface; the other being of Plexiglas reflects the ultrasound pulses incident upon it which then return to the transducer, again passing through the solution. The transducer, acting as an elastic wave receiver at this stage, is used to detect the reflected elastic wave, the physical characteristics of which (amplitude, phase, frequency spectrum) are obtained by analysing the electrical signal produced by the pass receiver.

The experimental procedure used first provides for filling the cylindrical cell with a buffer solution of Hepes (10 mM, pH 7.4) to obtain the reference acoustic signal; subsequently solutions of nanobubbles at different temperatures in the same Hepes buffer solution are placed in the cell to measure the attenuation of the echo of the reflected signal.

Figure 9 shows the change in attenuation (expressed as dB/cm) measured at the frequency of 14 MHz for the nanobubbles described here, as their concentration varies in Hepes buffer solution. A linear variation with increasing nanobubble concentration was found; the maximum attenuation value (approximately 6 dB/cm at a nanobubble concentration in Hepes of a little less than 40%) is comparable with that measured using the same technique in SonoVue^® which, as mentioned, is a commercially available ultrasound contrast agent formed of lipid microshells containing SF6.

The frequency spectrum of the ultrasound signal attenuation measured using the pulse technique, Figure 10, shows that the ultrasound frequencies at which elastic energy is most scattered by the nanobubbles in space increases with frequency for the same nanobubble concentration in Hepes, reaching maximum values in the frequency range between 22 and 24 MHz.

Sonoporation experiment results

Nanobubbles of Span 20/Chol and DMPC/Chol containing the fluorescent probe calcein prepared using the method according to the invention were used to carry out in vivo experiments to open up the blood-brain barrier (BBB) following exposure to focused ultrasound (FUS). The experimental conditions under which the investigations into passage across the BBB were carried out were developed using a series of prior experiments in which nanobubbles and Sonovue® were sonoporated in order to cross the BBB using a probe which normally does not cross it - Evans blue dye.

Each rat (totalling 5 per experiment) was anaesthetised with isoflurane and placed in a stereotaxic system. The focused ultrasound transducer was placed above the shaved head of the mouse, on which ultrasound gel was placed.

A suspension containing nanobubbles (0.13 μΐ/g for the experiment using DMPC/Chol nanobubbles and 0.08 μΐ/g for the experiment using Span 20/Chol nanobubbles, in concentrations of 2.14 x 10⁼⁸ and 1.28 x 10⁸ nanobubbles/ml) with gas and calcein within and the Evans blue probe present in the solution outside the nanobubble respectively was injected into the caudal vein of the rats (mean weight 30 g). FUS was then applied through a transducer with a frequency of 3.5 MHz, which was in turn connected to an ultrasound generator. FUS was applied for 55 seconds in a pulsed manner, with a 5 second pause, three times, in order to dissipate the heat produced by the ultrasound and to avoid overheating of the part.

The animals were sacrificed after 48 hours and ultrathin cryosections were made of the brains of every rat to observe the presence of fluorescence due to the presence of calcein carried into the brain via the nanobubbles, using fluorescence microscopy; the calcein itself was quantified using appropriate software (Image J software - https://imagej.nih.gov/ij/). The Evans blue probe, which is unable to cross the intact BBB in the absence of pores caused by Sonoporation, also passed into the brain following exposure to FUS.

Figure 13 shows quantification of the fluorescence in the brain following transport by DMPC/Chol and Span20/Chol nanobubbles. From the graph it is obvious that both the samples are efficiently capable of cavitating, thus releasing the calcein which will spread into the brain through the transient pores just created by the sonoporation. In this experiment, in which the quantity of calcein within the brain was not normalised with respect to the quantity transported by the nanobubbles, it was found that surfactant nanobubbles were more efficient at transporting calcein into the brain (Figure 13). The fact that both samples are in any event capable of carrying calcein across the BBB by sonoporation represents a finding of extreme interest in that it demonstrates the effectiveness of nanobubbles in the process of cavitation and sonoporation and joint transport of a hydrophilic and a gas molecule. This result demonstrates the applicability of such systems as targeting systems in the therapeutic field through the application of FUS externally to the site where the material trapped in the nanobubbles, diagnostic materials such as ultrasound and/or theranostic probes, have to be released.

References

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Claims

1. Nanobubbles comprising an external lipophilic double layer containing within it an aqueous layer located around a central gaseous portion.

2. Nanobubbles according to claim 1 in which the lipophilic double layer contains cholesterol in combination with at least one of phospholipids and surfactants selected from l,2-c palmitoyl-sn.-glycero- 3 -phosphocholine (DPPC), Span 20, l,2-chmyristoyl-sn.-glycero-3- phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), dicetyl phosphate (DCP), Span 60 and Tween 85, 81, 80, 65, 61, 60, 21, 20.

3. Nanobubbles according to either of claims 1 or 2 having a diameter of between 120 and 380 nm, preferably between 130 and 300 nm.

4. Nanobubbles according to any one of claims 1 to 3 having a stability in aqueous solution of at least 180 minutes at a temperature of between 37°C and 40°C.

5. Nanobubbles according to any one of claims 1 to 4 having a stability in aqueous solution of at least 3 months at a temperature of 4°C.

6. Nanobubbles according to any one of claims 1 to 5 in which the gas present in the central gaseous portion is selected from linear, branched or cyclic C3-C7 perfluorocarbons, preferably tetr adecafluorohexane .

7. Nanobubbles according to any one of claims 1 to 6, further comprising at least one agent selected from therapeutic and diagnostic agents of a lipophilic and hydrophilic nature and combinations thereof.

8. Nanobubbles according to claim 7 in which the agents of a hydrophilic nature are within the aqueous layer and the agents of a lipophilic nature are within the lipophilic double layer.

9. Nanobubbles according to any one of claims 1 to 8, further comprising at least one targeting molecule linked to the lipophilic double layer, preferably selected from polypeptides, polynucleotides, antibodies and antibody fragments.

10. Nanobubbles according to any one of claims 1 to 9 for use in the human, paediatric and animal medical, theranostic, diagnostic field, in particular ultrasound imaging.

11. Nanobubbles according to any one of claims 1 to 9 for use in treatments providing for a step of sonoporation of the blood-brain barrier.

12. Pharmaceutical compositions comprising nanobubbles according to any one of claims 1 to 9, and a pharmaceutically acceptable vehicle.

13. Pharmaceutical compositions according to claim 12 further comprising at least one agent selected from therapeutic and diagnostic agents of a hpophilic nature and hydrophilic nature and combinations thereof.

14. Composition according to either of claims 12 or 13 in which the polydispersity index of the dimension distribution curve for the nanobubbles in the composition is less than 0.25, preferably less than 0.20.

15. Composition according to any one of claims 12 to 14 for use in the human, paediatric and animal medical, theranostic, diagnostic field, in particular ultrasound imaging.

16. Composition according to any one of claims 12 to 14 for use in treatments providing for a step of sonoporation of the blood-brain barrier.

17. Method for the preparation of nanobubbles according to any one of claims from 1 to 9 comprising the following essential steps:

(a) preparing a film by dissolving an aliquot of cholesterol in combination with one or more of cationic phospholipids and surfactants in an organic solvent and subsequent evaporation of the said solvent;

(b) hydrating the film obtained in stage (a) with water;

(c) placing the mixture obtained in stage (b) in a sealed reactor, then adding an aliquot of a liquid perfluorocarbon in equilibrium with its gas phase;

(d) sonicating or extruding the suspension obtained in the previous stage at ambient temperature;

(e) cooling the suspension obtained in the previous stage without reaching the freezing point, preferably to a temperature of not less than 4°C.

18. Method according to claim 17 comprising a further step of purification of the suspension, preferably by centrifuging.

19. Method according to claim 18 in which centrifuging is performed at 600 rpm for 25 minutes at a temperature of 25° centigrade.

20. Method according to any one of claims 17-19 in which the perfluorocarbon has a linear, branched or cyclic C3-C7, preferably Ce, chain.

21. Method according to any one of claims 17-20 in which in step (a) an aliquot of an agent selected from lipophilic therapeutic and diagnostic agents and mixtures thereof is added to the organic solvent.

22. Method according to any one of claims 17-21 in which in step (b) water is completely or partially replaced by an aqueous solution containing an agent selected from hydrophilic therapeutic and diagnostic agents and mixtures thereof.

23. Method according to either of claims 21 or 22 in which the agent is selected from fluorescent, radioactive and luminescent probes.

24. Nanobubbles directly obtained with the process according to claims 17-23.

25. Nanobubbles directly obtained with the process according to claims 17-23 for use in the human, paediatric and animal diagnostic, and in particular ultrasound imaging and theranostic, medical fields.

Nanobubbles directly obtained with the process according to claims 17- 23, for use in treatments providing for a step of sonoporation of the blood-brain barrier