FIELD

The present invention relates to materials suitable for use as contrast agents in diagnostic imaging. The present invention further relates to ordered structures suitable for use in spectroscopy. The present invention also relates to methods of preparing the materials or ordered structures and methods of diagnostic imaging using the materials or ordered structures.

BACKGROUND

Nanoparticles are used in many applications such as delivery vehicles for therapeutic agents, optics, biomolecular separations and diagnostic imaging. Nanoparticles have shown particular use as contrast agents in a wide range of imaging techniques and in the case of Magnetic Resonance Imaging (MRI) magnetic nanoparticles are used to discern damaged tissues or organs from healthy tissues or organs.

Contrast agents are also used in other medical imaging techniques such as X-ray and fluorescent imaging. In these instances, the source radiation (eg X-ray or light) is diffracted, deflected or absorbed by the contrast agent particles. Different techniques for detecting the radiation transmitted through a contrast agent-containing sample and converting this into a diagnostic image, are known.

Whilst contrast agents greatly improve contrast in various medical imaging modalities, including MRI, X-ray, optical and nuclear, contrast agents still exhibit a number of limitations. One limitation relates .to ^ the amount of contrast agent particles that can be delivered to the area of diagnostic interest within a subject. A further limitation is the limited chemical or colloidal stability of the contrast agent particles in the medium within which it is delivered to the subject. There can be a tendency for contrast agents in the medium to aggregate rather than be evenly distributed. A further limitation is the chemical and biological stability of the contrast agent particles in vivo. A further limitation is the potential toxicity of many of the materials used in the fabrication of such contrast agent particles. Thus, it is of interest to deliver high concentrations of contrast agent particles in a medium within which they are stable, biocompatible and low in toxicity for a sufficient period of time and over a broad temperature range. Contrast agents used for other diagnostic imaging would also benefit from being presented in a format that is stable and biocompatible in the medium in which it is presented for a sufficient period of time, and with high loading of the signal generating species. Such systems would also be of interest for use in other fields of spectroscopy, and in fields that benefit from the distribution of high loadings of particles in a medium.

Thus, it is an object of the present invention to provide a new generation of improved contrast agents which allow an early diagnosis with high sensitivity as well as a differential diagnosis. The present invention relates to the incorporation of a high density of nanoparticles within a liquid crystal.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a material comprising: nanoparticles, amphiphile self-assembled into an ordered lyotropic liquid crystal phase, a liquid solvent in which the lyotropic liquid crystal phase is dispersed as nanodroplets, and a stabiliser for providing stabilisation against re- aggregation of the nanodroplets, wherein the nanoparticles are distributed within the dispersed nanodroplets of the lyotropic liquid crystal phase .

This material is particularly suited for use as a contrast agent.

Accordingly, in a second aspect of the invention there is provided a contrast agent comprising: nanoparticles, amphiphile self-assembled into an ordered lyotropic liquid crystal phase, a liquid solvent in which the lyotropic liquid crystal phase is dispersed as nanodroplets, and a stabiliser for providing stabilisation against re- aggregation of the nanodroplets, wherein the nanoparticles are distributed within the dispersed nanodroplets of the lyotropic liquid crystal phase .

In a third aspect of the invention the material, or contrast agent, is prepared by: i) mixing nanoparticles and a self-assembling amphiphile in a solvent; ii) removing solvent to provide a mixture of nanoparticles and the self-assembling amphiphile; iii) adding a liquid solvent to the mixture to form a self assembled lyotropic liquid crystal phase with nanoparticles distributed within the lyotropic liquid crystal phase and iv) adding a stabiliser and dispersing 'the lyotropic liquid crystal phase in the liquid solvent to produce dispersed nanodroplets of the lyotropic liquid crystal phase with nanoparticles distributed therein, the stabiliser providing stabilisation against re-aggregation of the nanodroplets.

In a forth aspect of the invention there is provided a liquid formulation comprising a liquid solvent containing a dispersion of nanodroplets and a stabiliser for maintaining the dispersion, wherein the nanodroplets contain nanoparticles distributed within an ordered lyotropic liquid crystal phase formed from a self- assembled amphiphile.

According to a further aspect of the invention there is provided the use of the material, contrast agent or liquid formulation according to the aspects of the invention described above as a diagnostic imaging agent, such as an MRI contrast agent.

According to an embodiment of the invention, there is provided a method of diagnostic imaging, the method comprising:

- administering to a subject a contrast agent, and - performing a diagnostic imaging procedure on the subject, wherein the contrast agent comprises: nanoparticles, amphiphile self-assembled into an ordered lyotropic liquid crystal phase, a liquid solvent in which the lyotropic liquid crystal phase is dispersed as nanodroplets, and a stabiliser for providing stabilisation against re- aggregation of the nanodroplets, wherein the nanoparticles are distributed within the dispersed nanodroplets of lyotropic liquid crystal phase. _ c _

It has been found that in each of these aspects high loadings of nanoparticles are achieved within the lyotropic liquid crystal phase or matrix. It is also found that the nanoparticles are loaded into the lyotropic liquid crystal phase with a high degree of regularity, density or clustering. This results from the nano-scale porosity of the self assembled matrix, which provides a high internal and external surface area. The nanoparticles are distributed within a region of the nanostructure that is compatible with the nanoparticle surface (i.e. hydrophobic surfaced nanoparticles will report to a hydrophobic region, or hydrophilic to a hydrophilic or aqueous region) , so they too are distributed within the discrete nanostructures in an ordered or random arrangement, and at high concentration due to the large internal and external liquid crystal surface area.

In addition, these materials have high chemical, colloidal and biological stability through the appropriate selection of the lyotropic liquid crystal phase-forming materials.

These materials are also biocompatible through the appropriate selection of the lyotropic liquid crystal phase forming materials.

In addition, these materials show reduced toxicity relative to the nanoparticles alone by the encapsulation of the nanoparticles within the liquid crystal matrix, which precludes direct exposure of the nanoparticles to the subject.

Furthermore this arrangement provides an ideal environment for the delivery of contrast agents that will enhance diagnostic imaging techniques, such as MRI, that benefit from the high concentrations of contrast agent particles.

In one embodiment of the invention the material contains at least two different types of nanoparticles (that is, nanoparticles of different chemical compositions) which offer multimodal imaging capabilities.

According to another embodiment of the invention, the nanoparticles have magnetic, paramagnetic or superparamagnetic properties, for example iron oxide nanoparticles. Such nanoparticle materials are suitable for specific types of medical imaging, as described in further detail below.

Preferably the nanoparticles are coated. More preferably the coating imparts a hydrophobic surface to the particle. This facilitates the insertion, packing or distribution and retention of nanoparticles into the lyotropic liquid crystal phase.

In another embodiment, the concentration of nanoparticles in the solution of nanodroplets of the lyotropic liquid crystal phase dispersed in the liquid solvent, is relatively high. In the case of iron oxide nanoparticles, the concentration of particles (represented by the concentration of iron) in the solution of nanodroplets dispersed in the liquid solvent is between 1 to 10 mmol/L, preferably between 1 to 5 mmol/L.

In another embodiment of the invention, the average number of nanoparticles per nanodroplet is between 1 to 300, preferably between 10 to 150, more preferably between 50 to 100.

According to another embodiment of the invention, the nanoparticles are distributed within the nanodroplets in clusters, with the number of clusters of nanoparticles within the nanodroplets preferably between 1 to 30, and more preferably between 1 to 15. Preferably, _' the number of nanoparticles within each cluster is between 1 to 150, more preferably between 10 to 100.

In another embodiment of the invention, the nanodroplets have an average diameter of less than 1000 nm, preferably less than 500 nm, more preferably between 50 and 550 nm, particularly between 100 and 200 nm.

Preferably the lyotropic liquid crystal phase is a cubic phase. The cubic phase is preferred due to the nature of its ordered internal nanostructure, which accommodates the nanoparticles in a preferred arrangement.

For the materials and methods described above, the product is in the form of discrete nanodroplets of the lyotropic liquid crystal matrix which contains distributed nanoparticles within. According to an alternative aspect suited to other applications, such as separation of nanoparticles from media, the lyotropic liquid crystal matrix containing the distributed nanoparticles is retained as a bulk phase, and is not dispersed into discrete nanodroplets.

According to this embodiment, there is provided a material comprising: nanoparticles, a matrix comprising amphiphile self-assembled into an inverse cubic phase matrix, wherein the nanoparticles are distributed within the inverse cubic phase matrix.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Nanoparticles

The material of the present invention contains nanoparticles distributed within a lyotropic liquid crystal _' or liquid crystalline phase> or matrix. Nanoparticles are particles of a size with at least one dimension less than 50nm. Preferably, the nanoparticles used in the present application are not greater than 120nm in any dimension. More preferably, the nanoparticles have an average particle size of between 1 and 30nm, particularly between 1 and 15nm.

The nanoparticles may be formed from any suitable organic or inorganic material. For applications where the nanoparticles are functioning as contrast agents, the nanoparticles are formed from a material which generates contrast for a given imaging modality - that is, a contrast-generating material. The contrast generating material is a material that interacts with incident radiation or fields and causes a modification to the transmitted, reflected, scattered or absorbed signals. However, where the nanoparticles are serving other functions not related to medical imaging, they may be of any other suitable constitution for the intended application.

The nanoparticles may therefore be formed from a material selected from one or more of metals, metal oxides (including magnetic metal oxides) , metal hydroxides, metal complexes, metal sulphides, metal selenides, metal carbides, carbonaceous materials (such as polymers, dendrimers, proteins, peptides, nucleic acid, virions, lipids, oils, carbon nanotubes and peptidyl nanotubes) core-shell particles (as in the case of microbubbles, such as Levovist™ and Optison™ used in ultrasound) and quantum dots(qdots), gold and inorganic nanoparticles.

Within this class are the types of materials used as contrast agents for diagnostic imaging. These are described in further detail below with respect to particular imaging types. According to some embodiments, the nanoparticles are formed from contrast agent materials. Contrast agent materials are materials that provide a contrast when subjected to a diagnostic imaging procedure, such as fluorescence imaging, x-ray, magnetic resonance imaging, and so forth. Examples of contrast agent materials include magnetic materials, fluorescent materials and x-ray detectable materials. One subclass of interest are magnetic, paramagnetic or super-paramagnetic nanoparticles .

Contrast agents for MRI are typically magnetic materials that enhance the relaxation time. MR contrast agents may be either positive agents (also referred to hereinafter as "Tl agents") that illuminate or "light up" the tissue that they occupy, or negative agents (also referred to hereinafter as "T2 agents") that make a tissue appear darker. Positive Tl agents have a relaxivity Rl, where R1=1/T1, whereas negative T2 agents have a relaxivity R2, where R2=l/T2. Examples of Tl agents include, but are not limited to paramagnetic gadolinium complexes (metal complexes), such as Gd-DPTA and the like. Non-limiting examples of T2 agents include superparamagnetic iron oxide (SPIO) nanoparticles. Superparamagnetic nanoparticles provide higher relaxivities than paramagnetic agents, as they generally have magnetic moments that are about 100 times greater than those of paramagnetic agents.

A particular sub-class of interest are nanoparticles which predominantly show MRI-T2 contrast. Examples of MRI-T2 contrast materials are the iron oxides and mixed oxides of iron with other metals, and mixtures thereof. Examples are hematite (Fe ₂ O ₃ ), ferrite (Fe ₃ O^ and magnetite, which is a mixed spinel ferrite having the general formula MFe2U4 where M is a metal such as, but not limited to, manganese, cobalt, copper, nickel, gadolinium, zinc and vanadium; and combinations thereof.

Fluorescence contrast agents may include fluorescent nanocrystals such as quantum dots, photoproteins, such as fluorescent green protein, porphyrin, fluorescamin or ribolabin. X-ray contrast agents include iodinated media such as iothalamate diatrizoate, ioxaglate, non- ionic monomers such as iopromide or non- ionic dimers such as iotrolan.

Liquid crystals

Liquid crystals (LC) are substances that exhibit a phase of matter that has the intermediate properties of a conventional liquid, and those of a solid crystal.

Lyotropic LCs exhibit phase transitions as a function of concentration of the amphiphile in a solvent (typically water) as well as temperature.

Lyotropic liquid crystal phase

A lyotropic liquid crystal phase consists of two or more components (generally amphiphile and solvent) that exhibit liquid-crystalline properties within a specific concentration range. The phase behaviour of lyotropic liquid crystals is dependent on both temperature and concentration with respect to solvent (such as water) and other environmental factors such as pressure. In general, a lyotropic liquid crystal phase comprises amphiphile and a solvent (usually water) . Amphiphiles are a class of compounds comprising an immiscible hydrophilic and hydrophobic region. The assembly of the amphiphiles results in a number of distinct phases and structures of varying complexity or dimensionality. The phases include the lamellar phase (La) (1-D) , which is comprised of sheets of stacked bilayer sheets, the hexagonal phase (H ₁₁ ) (2-D), which can be conceptualised as infinitely packed cylindrical rods with an aqueous interior, or the cubic phase, which can be conceptualised as a 3 dimensional interpenetrating network of hydrophilic and hydrophobic domains. When the cubic phase is dispersed into small particles or nanodroplets retaining the cubic phase structure, these particles or nanodroplets are sometimes termed "cubosomes". The structures may also include spherical micelles and other intermediate phases.

The term "lyotropic liquid crystal phase" is used herein to refer to the structure of the material that forms part of the contrast agent. The lyotropic liquid crystal phase of the present invention is formed from amphiphiles which self assemble, and thus the term excludes so-called

"ceramic glasses". The lyotropic liquid crystal phase or matrix is characterized by nanoscale domains which are clearly distinguished from neighboring domains by large differences in local chemical composition, and do not include materials in which neighboring domains have essentially the same local chemical composition and differ only in lattice orientation. Thus, by the term 'domain' as used herein, is meant a spatial region which is characterized by a particular chemical makeup which is clearly distinguishable from that of neighbouring domains: often such a domain is hydrophilic (hydrophobic) which contrasts with the hydrophobicity (hydrophilicity) of neighbouring domains. In the context of this invention the characteristic size of these domains is in the nanometre range.

Anderson et al (US Patent No. 6,638,621 B2 ) is incorporated herein by reference to provide examples of the lyotropic liquid crystal phases that can be used in the practice of the present invention.

Amphiphiles and phase behaviour

The lyotropic liquid crystal phase of the present invention is created by the self-assembly of amphiphiles. Examples of amphiphiles are lipids, surfactants (synthetic or otherwise), polymers, block co-polymers, and mixtures thereof. The packing of each amphiphilic molecule can be described by the critical packing parameter, S, which is described by the relationship S = v (αol _c ) , where v is the molecular volume of the chains, l _c is the molecular length of the chains and cto is the optimal head-group area for each amphiphile monomer (See Figure 1) . When the packing parameter is less than unity (S < 1), the resulting aggregate is curved towards the hydrophobic region as in normal micellar aggregates (Li) , while for molecules where S « 1, a cylindrical molecular geometry is suggested, resulting in the formation of a flat bilayer (lamellar phase) . If the amphiphile contains a hydrophobic moiety that occupies a large volume and correspondingly small head-group, then S > 1, and the aggregate is curved towards the polar region, as observed with cubic or inverse hexagonal phases.

In the context of the present invention, amphiphiles with a critical packing parameter (S) of H to >1 are preferred, further preferably H to >1.

The hydrophilic-lipophilic balance (HLB) is a measure of the hydrophilicity or lipophilicity of a molecule by use of the formula HLB = 20 * Mh / M where Mh is the molecular mass of the hydrophilic portion of the Molecule, and M is the molecular mass of the whole molecule. This provides a result on an arbitrary scale of 0 to 20, where an HLB value of 0 corresponds to a completely hydrophobic molecule, and an HLB value of 20 would correspond to a molecule made up completely of hydrophilic components. HLB can be used as a parameter to determine the suitability of an amphiphile for use in the present invention. Preferably, an amphiphile of the present invention has an HLB of at least 1, more preferably between 1-10.

A variety' of■ geometries that form .through .the self- assembly of amphiphiles as described above are envisaged. Some examples include inverse micellar, inverse hexagonal, lamellar, inverse cubic, bicontinuous cubic, normal cubic, normal hexagonal, micellar, among others.

Of particular interest is the inverse cubic phase in which an amphiphile bi-layer is folded onto an infinitely periodic minimal surface (IPMS) consisting of infinite interwoven arrays of saddle surfaces with zero mean curvature and constant negative Gaussian curvature. The middle of the bilayer, defined by the terminal methyl groups of the intersecting hydrophobic moieties, forms the IPMS and the hydrophilic parts interface with the bulk water (Figure 2). A surfactant bilayer (1) and an aqueous water channel (2) of an inverse cubic phase is shown in Figure 2.

This molecular architecture affords distinct aqueous regions that form two continuous water networks (or channels) throughout the cubic phase. Long-range crystallographic periodicity is found in such structures, with crystallographic space groups including the Im3m, Pn3m and Ia3d (Figure 3) . Each of these space groups differs in water content and tortuosity, conferring different properties to each.

The inverse cubic liquid crystal phase is thermodynamically stable and co-exists in equilibrium with excess water over a broad temperature range.

An inverse cubic liquid crystal phase provides an appropriate scaffold in which to distribute or package up nanoparticles in a biomimetic environment. In addition, owing to the high surface area of the internal liquid crystal structure (up to 1000 m ² /g, such as up to 400 m ² /g) , the inverse cubic phase affords a high concentration of nanoparticles in a three dimensional ordered array , which can be of benefit to at least diagnostic imaging. Synthesis of the Contrast Agent

In the preparation of the material or contrast agent the nanoparticles and the self assembled amphiphiles are combined to prepare the contrast agent.

Synthesis of nanoparticles

The nanoparticles may be hydrophilic or hydrophobic or they may be treated to change their hydrophobicity . The choice of solvent used to prepare the agents is made depending on whether the nanoparticles are to be hydrophobic or not.

In general the preparation of the nanoparticles for incorporation into the liquid crystalline phase involves nucleation of nanoparticles from a precursor solution. The solvent can be aqueous or organic, however, organic solvents are preferred as they allow greater control of reaction conditions such as temperature and hence greater control over the growth and particle size of the nanoparticles. A non-aqueous solvent can also be chosen such as an ionic liquid, DMSO and so forth, and the resulting nanoparticles will impart a hydrophilic nature.

Coating of nanoparticles

According to one preferred embodiment the nanoparticles have a hydrophobic surface before they are mixed into the amphiphile. The nanoparticles may have a native hydrophobic surface, or may be subjected to a treatment (such as coating or other modification by surface attachment of pendant hydrophobic groups through chemisorption or physisorption) to induce a hydrophobic surface. The treatment may be constituted by a step in the preparation of the nanoparticles.

In the context of coatings, suitable coating agents comprise distinct hydrophobic and hydrophilic region. Thus, the coating agents may suitably be selected from amphiphiles such as anionic, cationic, non-ionic, zwitterionic surfactants, fatty acids, alkanethiols, alkylsilanes, polymers (including block co-polymers, biopolymers, polyethylene oxide and polyacrylamide as examples) . Amongst the fatty acids that may be used are fatty acids with a carbon chain length of C4-C ₂₈ , such as oleic acid. Other suitable surfactants include oleylamine and trioctylphosphine (TOPO), as examples.

In the final material or contrast agent, the nanoparticles remain coated by the coating material (such as surfactant) , thus providing a hydrophobic surface for the nanoparticles. In the preparation of iron oxide nanoparticles, a reducing agent is also added to partially reduce the iron to form the required magnetic oxide. One example of a reducing agent is a long chain hydrocarbon diol .

Alternatively if the nanoparticles are to be distributed within an aqueous part of the lyotropic liquid crystal phase, then it may be necessary to make the nanoparticles hydrophilic. This can be achieved by exchanging existing ligands or over-coating the nanoparticle with a suitable hydrophilic stabiliser.

Distribution of nanoparticles into the lyotropic liquid crystal phase

In one aspect the nanoparticles are distributed within a lyotropic liquid crystal phase. The distribution is preferably one of high density. By distribution we mean a packaging of the nanoparticles within the internal structure of the lyotropic liquid crystal phase, as opposed to superficial encapsulation.

To distribute the nanoparticles within the lyotropic liquid crystal phase, the nanoparticles and the amphiphile are mixed in a first solvent, this first solvent is then removed and a second solvent is added to swell/hydrate the mixture into a matrix which is gel like in nature. The matrix formed on swelling/hydration by the second solvent is a continuous volume of lyotropic liquid crystal phase.

Dispersion of the lyotropic liquid crystal

Once the nanoparticles are distributed within the lyotropic liquid crystal phase, a further dispersion occurs to divide the continuous volume of lyotropic liquid crystal phase into "nanodroplets" of this matrix. This dispersion can be achieved by any method known in the art, typically high pressure homogenisation or extrusion.

Dispersion into nanodroplets is necessary to reduce the viscosity of the material, to disperse the material in a solvent that is biocompatible, and to disperse the material in a solvent that is compatible for introduction into the subject (e.g. subcutaneous injection) .

The final material or contrast agent which has nanoparticles distributed within a dispersed lyotropic liquid crystal phase is itself a dispersion of nanodroplets or "particles" of the lyotropic liquid crystal. The distribution of the nanoparticles is not necessarily regular, ordered or uniform. It may be random. The nanoparticles may form arrays or clustered, or concentrated in particular channels of the final nanodroplets. They may concentrate around the circumference of the final nanodroplet, or within a certain layer which may also be circumferentially bound.

The reference to nano in the context of the nanodroplets refers to a droplet (particle) diameter of less than 1000 nanometers, preferably less than 500 nm.

The size range of such dispersed lyotropic liquid crystal nanodroplets (or particles) is suitably between 50 - 550 nm in diameter. Typically the size range is around 150nm, +/- 50nm.

A stabiliser is required to stabilise the nanodroplets of lyotropic liquid crystalline phase material loaded with nanoparticles to prevent aggregation or re-aggregation. The stabilizer is an agent that prevents aggregation, coalescence and flocculation of dispersed lyotropic liquid crystal phase particles. Stabilisers impart colloidal stability to the dispersion. Suitable stabilisers include agents such as small particulates that are capable of being adsorbed upon surfaces of the nanoparticles, ionic materials, polymers (including copolymers, block copolymers, etc) , proteins, charged lipids, surfactants, and liquid crystalline phases adsorbed to the surface of the particles. Block copolymer stabilisers such as the Pluronic range of stabilisers and their equivalents are notable examples. Pluronic encompasses a wide range of block copolymer surfactants, typically based on ethylene oxide and propylene oxide, marketed by BASF. These are non-ionic surfactants. Other classes of non-ionic surfactants may be used.

Imaging

One advantage of the contrast agent medium is that by appropriate selection of the nanoparticles, the contrast agent is detectable by more than one different imaging technique. The contrast agent in this case is a contrast agent medium for two or more modes of diagnostic imaging or in other words is a multimodal contrast agent. In the method of diagnostic imaging, the method preferably comprises the step of performing the diagnostic imaging on the subject using at least two different diagnostic imaging modes utilising the multimodal contrast agent. BRIEF DESCRIPTION OF DRAWINGS:

Fig 1 is a schematic representation showing the various liquid crystalline phases of lipids as described by the critical packing parameter S, defined by S = V (Ot ₀ Ic) ^'1 wherein V is the molecular volume of the chains, Ic is the molecular length of the chains and α _o is the optimal head- group area for each amphiphile monomer.

Fig 2 is a 3-D representation of the (inverse) cubic phase.

Fig 3 shows a series of 3-D representations of the crystallographic space groups of the (inverse) cubic phase of (1) Im3m, (2) Pn3m and (3) Ia3d.

Fig 4 is a schematic representation showing the production of dispersed bulk lyotropic liquid crystal using high pressure homogenization according to an embodiment of the invention.

Fig 5 is a cryo-TEM image of an embodiment of the invention showing iron oxide nanoparticles in a phytantriol derived cubosome stabilised with pluronic F- 127.

Fig 6 is a cryo TEM image showing embodiments of the invention of 15 nm spherical SPIOs in cubosomes.

Fig 7 is a cryo TEM image showing embodiments of the invention of 15 nm spherical SPIOs in cubosomes.

Fig 8 is a cryo TEM image showing embodiments of the invention of 8 nm spherical SPIOs in a cubosome.

Fig 9 is a cryo-TEM image showing an embodiment- of ^; the invention of gold nanoparticles in a phytantriol and pluronic F- 127 ,

Fig 10 is a cryo TEM image showing an embodiment of the invention of qdot and SPIO incorporation into cubosomes.

Fig 11 is a cryo TEM image showing an embodiment of the invention of protein stabilised cubosomes.

Fig 12 is a histogram comparing Cubosome size stabilised with either pluronic F127 or protein (sodium caseinate) according to embodiments of the invention.

Fig 13 is a plot of T2 Relaxivity for various SPIO cubosome formulations according to embodiments of the invention compared to SPIO in water.

Fig 14 is an X-ray phase contrast image of cubosome-SPIO nanoparticles according to an embodiment of the invention injected inside mouse brains.

Fig 15 (a) and (b) are MRI Tl and T2 scans in blood samples of a cubosome (composed of phytantriol and pluronic F-127, into which iron oxide has been dispersed) according to an embodiment of the invention compared with Gadolinium (Gd) and water, and pure blood as a control.

Figure 16 are MRI scans carried out with nanodroplets according to an embodiment of the invention that were injected into a mouse.

EXAMPLES

The invention will now be described with reference to the following non-limiting examples.

Example 1

Preparation of 'Lyotropic Liquid crystal phase' : Iron-oxide nanoparticles distributed in lyotropic liquid crystal phase composed of the amphiphile phytantriol (3,7, 11, 15-tetramethyl-l, 2, 3-hexadecane-triol) and Pluronic F-127, and dispersed into an aqueous matrix:

i) Preparation of nanoparticles

2mmol of Fe(acac)3, 10 mmol of 1,2 hexadecanediol, 6 mmol of oleic acid and 6 mmol of oleylamine was dissolved into 20 mL of benzyl ether, and, using traditional Schlenk line techniques, was equilibrated at 200 °C for 2 hours under nitrogen then heated to reflux at 300 ⁰ C for a further hour. The mixture was then allowed to cool to room temperature. A black material was precipitated from the mixture by adding ethanol and separated via centrifugation. The solids were then dissolved in hexane containing 1 drop each of oleic acid and oleylamine, and the undispersed residue was removed by centrifugation (6000 rpm, 10 mins) . The nanoparticles were then precipitated from the concentrate with ethanol, centrifuged to remove the solvent and redispersed in another solvent - hexane.

ii) Treatment of nanoparticles At this point the particles produced in step i) are already coated in oleic acid so there are no other steps required to make the particles hydrophobic.

iii) Choice of amphiphile The surfactant 3, 7, 11, 15-tetramethyl-, 1, 2, 3- hexadecane-triol or ^λ phytantriol' is employed as the amphiphile in this example. It swells into a bicontinuous cubic phase of the inverse type. It exists over a broad temperature range and is not susceptible to hydrolytic degradation that other surfactants such as glycerol mono olein (GMO) may undergo . phytantriol

iv) Process steps of introducing the treated nanoparticles to the amphiphile

A stock solution of the iron oxide nanoparticles in hexane, was prepared as described above in i) and ii) . A 3 rtiL aliquot of the solution was added to 1 g of pure phytantriol, followed by the addition of 5 mL of dichloromethane to ensure complete solubilization of the phytantriol in the solvent system. Once all components of the mixture were dissolved, the solvent was removed under reduced pressure to facilitate transfer of the nanoparticles into the phytantriol.

v) Dispersion

A dispersed lyotropic liquid crystal or matrix was prepared using nanoparticle-functionalized surfactant of step iv) . One gram of nanoparticle- surfactant mixture was placed in a small glass beaker. The stabiliser Pluronic F-127 (.07 g) was added, and the beaker was placed in an 80° C oven for three minutes. The contents were stirred with a metal spatula until all Pluronic dissolved completely. The mixture was then slowly dripped into a plastic jar containing 29 mL of water (warmed in an oven to 80° C) while being stirred by the heated (80° C) arm of a Polytron high shear mixer. The solution was mixed for five minutes, then poured into the cylinder of an Avestin high pressure homogenizer in a water bath heated to 60° C. The solution was run through the homogenizer five times with the pressure varied manually to produce a drip rate of about one drop per second (usually between 40 and 60 psi on the pressure gauge) . During each run, the solution was collected in the plastic jar held submerged in the water bath. After the final run the particle size distribution for the nanodroplets was obtained using a Coulter LS Variable Speed Fluid Module Plus particle size analyzer.

vi) Analysis of the products

Three key techniques are used in the characterization of the lyotropic liquid crystal phase and the intermediate materials. These include : • Light Polarizing microscopy;

• Small Angle X-ray Scattering (SAXS) ; and

• Cryogenic Transmission Electron Microscopy (Cryo- TEM) .

Light Polarizing Microscopy is a technique used to qualitatively identify the phases which exist over a broad temperature range and in excess water. This technique characterises the phase behaviour of the bulk gel material and not the dispersions. Small Angle X-ray Scattering (SAXS) is employed to quantitatively identify the phases present in a lyotropic liquid crystal sample (for bulk liquid crystal and dispersion) . Cryogenic Transmission Electron Microscopy (Cryo-TEM) is an imaging technique used to characterise the dispersions. Representative Cryo- TEM images of the various nanoparticle/phytantriol DLM materials are shown below (Figures 5-11). The Cryo-TEM confirms the preparation of the dispersed lyotropic liquid crystal nanodroplets, the internal ordering of the dispersed lyotropic liquid crystal, the mean particle size and distribution, the location of the added nanoparticles within the dispersed lyotropic liquid crystal, and the ordering of the added nanoparticles within the dispersed lyotropic liquid crystal.

In particular, Figure 5 shows ordered arrays, regular clusters, of cubic SPIOs produced within the cubosome materials. The large cubosomes measure 260 and 275 in diameter. Figure 6 shows 15 nm spherical SPIOs distributed either throughout the material but in ordered clusters or arrays or distributed around the circumference or the inner boundary of material. Figure 7 shows 15 nm spherical SPIOs in cubosomes that are irregularly dispersed, but the distribution is throughout the material. Figure 8 shows 8 nm spherical SPIOs in a cubosome measuring 120 nm in diameter. In this case there is a high density and the random distribution of the SPIOs throughout the material.

Example 2: Nanoparticles of gold.

Material was made according to Experiment 1, with the exception that gold was used instead of iron oxide.

Typically, 15mL of 0.03M hydrogen tetrachloro aurate (III) solution in water was added to 4OmL 0.05M tetraoctylammonium bromide solution in toluene while stirring vigorously. The transfer of gold precursor from water into toluene completed when the organic phase was a deep red wine colour while the aqueous was colourless.

The ligand solution, 0.45 mmol of 1-dodecanethiol (DDT) in toluene was then added to the organic phase. A freshly prepared aqueous solution of 5mmol sodium borohydride was added slowly to the reaction mixture with vigorous stirring. On addition of the reducing agent, the organic phase changed from an orange colour to a deep brown colloidal solution. After further stirring for 3 hours, the organic phase was separated and rotary evaporated to about 10 mL. 400 mL of ethanol was then added to remove excess thiol. The mixture was kept at -18 ⁰ C for 4 hours and the dark brown precipitate was recovered via centrifugation. The product was washed by redissolving in 1OmL of toluene, again precipitated with 400 mL ethanol and recovered via centrifugation, dried and weighed. The final gold nanocrystals were made up to a concentration of 0.02 M in toluene. High-resolution TEM image of the DDT stabilized nanoparticles revealed particle diameters within the range of 1-3 nm and a maximum in particle size distribution at 2.0-2.2 nm. Phase transfer into the bulk cubic phase and subsequent dispersion was achieved via experiment 1.

The material was characterised using Cryo TEM and the resulting images are displayed in Figure 9.

Example 3: Multimodal cubosomes containing qdots and SPIOs:

Cubosomes loaded with SPIOs and quantum-dots (qdots) have been prepared as per Example 1. Two q-dots emitting at 577nm, Extinction Coef: 1.96E+04 L/mol/cm, 4.7nm diameter and another emitting at 521nm, Extinction Coef: 1.45E+04 L/mol/cm, 5.3nm were used. The qdot samples were loaded into cubosomes using the method described in Experiment l(v) . Appropriate amounts of Q-dots (2mM) , SPIOs (2mM) and phytantriol were dispersed in chloroform and excess solvent was evaporated. Figure 10, shows qdots (and SPIOs loaded into cubosomes. The cubosomes (form of nanodroplets produced by the liquid crystal cubic phase) shown in Figure 10 have an average diameter in the range from approximately 40 nm to 125 nm. The SPIOs in the cubosomes are represented by the larger dark black globules that have a diameter of about 9-13 nm and the Q-dots in the cubosomes are represented by the smaller black globules having a diameter of about 5-6 nm.

Cryo TEM images of the qdot and SPIO incorporated into cubosomes were taken and revealed the presence of both imaging agents within the same cubosomes in some, cases, see Figure 10. Example 4: Stabilisation of cubosomes with proteins Bare, unloaded cubosomes have been produced from phytantriol (concentration, dispersed as per Example 1 (v) ) using sodium caseinate as a stabiliser instead of

F127. Figure 11 shows an image of a cubosome produced with this protein having an average diameter of about 120 nm. Note the absence of aggregated cubosomes. 1 g of sodium caseinate was solubilized in 30 ml of water. 1 g of hot phytantriol was added dropwise to hot sodium caseinate solution while applying shear homogenization. Then the dispersion was further processed using high pressure homogenisation (Avestin) . The temperature of the prepared solutions and processing times were prepared following the protocols from example 1 (v) .

A direct comparison was then made between the protein and the polymer, Pluronic F127 stabiliser and a range of concentrations were explored at the same time. Figure 12 illustrates the trend that the higher the concentration of the stabiliser (be it protein or surfactant) the smaller the cubosome diameter.

Example 5. MRI performance of SPIO loaded cubosomes Three different types of SPIO nanoparticles were loaded into cubosomes at various concentrations, using the methodology outlined in Experiment 1. Two were spherical monodisperse SPIOs of 8nm and 15nm diameter and the third was a cubic particle 9nm in size. The MRI performance was assessed in multiwall plates inside a clinical MRI machine. The data is summarised in Table 1 and plotted in Figure 13.

In all cases a significant enhancement of T2 relaxivity was seen when the nanoparticles were incorporated into the cubosomes. This probably indicates, an enhancement of the SPIO signal due to an increase in the rotational correlation constant of the SPIOs in cubosomes compared to the SPIO in free solution (polymer stabilised) . The T2 relaxivity of the 9nm cubic nanoparticles can be doubled (200% improvement) when they are incorporated into the cubosomes, while the signal of the 8nm spherical nanoparticles was enhanced 4.7 times (470%) at an iron concentration of 2865μM in the cubosome stock solution (g/ml phytantriol and g/ml of pluronic F127) . This is an extremely significant result as it allows much less iron to be injected for the same signal enhancement. The larger spherical iron oxide nanoparticles (15nm) also showed enhanced T2 relaxation rate compared to free solution, although the difference was not as great as the differences shown for the smaller nanoparticles.

These results were compared to Resovist which is well used imaging agent. The results of these novel cubosomes demonstrate comparable relaxivities to Resovist, which infers that the materials would be good imaging agents.

Table 1: Iron, phytantriol and pluronic concentration of each cubosome batch and the measured T2 relaxivity at each iron loading.

In Table 1 above, the concentration of iron present as iron oxide nanoparticles in the cubosome solution (i.e. the solution of nanodroplets of the lyotropic liquid crystal phase dispersed in the liquid solvent) was between about 1 to about 10 mmol/L, more typically between 1 to 5 mmol/L. The concentration of the amphiphile was between about 0.03 to about 0.06 g of amphiphile per ml of solvent. The stabiliser to amphiphile ratio, by weight, was about 0.1:1 to 0.2:1.

Example 6.: X-ray phase contrast of SPIO loaded cubosomes Cubosomes prepared according to Experiment 1, being loaded with SPIOs (2865μM) stabilised with F127 were injected into the fixed brains of mice and then X-ray phase contrast imaging was performed through the formalin fixed brains. The X-ray images of Figure 14 illustrate that the contrast agents of this invention can be detected in X- rays which infers their appropriateness as a contrast agent. In Figure 14, the arrows point to the white regions showing good X-ray phase contrast, with the image on the lower left showing the sectioned mouse brains.

Example 7: MRI Studies IN VITRO:

In vitro studies were undertaken on a Bruker Biospec 4.7 Tesla animal MRI scanner. Blood samples were subjected to Magnetic Resonance Imaging (MRI) using the iron oxide/ dispersed lyotropic liquid crystal systems as produced in the above examples. Representative images of the MRI scans are shown in Figure 15. The MRI scans show the results for Tl and T2 scans (positive and negative contrast, respectively) . The iron oxide/phytantriol dispersed lyotropic 'liquid crystal were compared with Gadolinium (Gd) and water. The contrast of pure blood was also measured as a control. From the scan shown in Figure 15a, the liquid crystal systems displayed minimal Tl contrast, as shown by the absence of a white image. The liquid crystals did, however, show a very good T2 performance, as noted by the very dark sample image (Figure 15b) .

IN-VIVO:

MRI scans were carried out with contrast agents comprising dispersed, nanoparticle-loaded liquid crystal nanodroplets as described above injected into a mouse. A cubosome solution containing 0.7 mM of Fe in cubosomes in ImI of PBS was injected into the femoral vein of an anaesthetised mouse and images were taken 5 minutes post injection. The results for Tl (not shown) and T2 (Figure 16) scans are similar to the blood samples, the contrast agent displayed minimal Tl contrast, shown by an absence of a white image. In Figure 16, the ROI locations refer to the regions of interest. Excellent T2 performance was measured for the systems. Furthermore, the results indicate that the nanodroplet particles (of liquid crystals) are rapidly cleared to the liver, suggesting that site specific targeting of the systems is critical.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.