Nuclear magnetic resonance contrast agent

Abstract

A contrast agent composition comprising outer shells, each outer shell enclosing an outer filling that comprises a dispersion medium with one or more inner shell(s) dispersed in the dispersion medium. The inner shell(s) each enclose an inner filling that comprises at least one contrast agent. The inner filling has one property and the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic. At least one of the outer shell and the inner shell is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively. The contrast agent composition can be used in nuclear magnetic resonance imaging.

Description

[0001] Nuclear magnetic resonance contrast agent

[0002] Description

[0003] Field of the invention

[0004] The invention relates to a contrast agent composition. It also relates to uses of the contrast agent composition. The invention moreover relates to a method of medical imaging. Further, the invention relates to methods of manufacturing a contrast agent composition.

[0005] Background of the invention

[0006] In many diagnostic procedures that involve nuclear magnetic resonance imaging (MRI), such as tumour characterisation, the use of a contrast agent is essential. Yet, recently the European Medicines Agency and the US Food & Drug Administration warned against a particularly common type of contrast agent used in MRI angiographic and perfusion diagnostic procedures, Gadolinium (Gd)-bearing contrast agents (GBCAs). These decisions were prompted by potential health concerns resulting from long-term accumulation of Gd- containing species in in various organs, including the brain. Accordingly, there is a need for MRI contrast agents for angiographic and perfusion diagnostic procedures, which contrast agents avoid the health risks of GBCAs.

[0007] The search for alternatives to Gd-based contrast agents for angiographic and perfusion MRI examinations led to development of the techniques for hyperpolarization of water protons via dissolution Dynamic Nuclear Polarization (dDNP), as for example described in Pinon et al, “Hyperpolarized water through dissolution dynamic nuclear polarization with UV-generated radicals”, Communications Chemistry, 2020, 3:57, https: / / doi.org / 10.1038 / s42004-020-0301- 6. However, the preparation of hyperpolarizing water protons is challenging as it requires a chain of sample preparation steps which include removing the paramagnetic molecular oxygen from the sample, reducing the proton concentration by mixing the sample with controllable amount of D2O, and keeping the sample at elevated temperature. Other alternatives to Gd-based contrast agents include the use the2H (deuterium) metabolic imaging (DMI) and hyperpolarized13C metabolic MRI (13C-HPMRI) which are emerging techniques to probe dynamic changes in tissue metabolism using MRI without the use of ionizing radiation, see for example Vaeggemose et al, “Comprehensive literature review of hyperpolarized carbon-13 MRI: the road to clinical application”, Metabolites 11 , 2021 , 219, and Woitek et al, “The use of hyperpolarised 13 C-MRI in imaging to probe cancer metabolism”, Br J Cancer 124, 2021 , 1187-1198. DM I is most frequently undertaken with oral deuterated [6,6'-2H2] glucose to detect the subsequent formation of its down-stream metabolites, see De Feyter et al, “Deuterium metabolic imaging (DMI) for MRI-based 3D mapping of metabolism in vivo”, Sci Adv 4, 2018, eaat7314, and de Graaf et al, “On the magnetic field dependence of deuterium metabolic imaging”, NMR Biomed. 33, 2020,e4235 .

[0008] Object of the invention

[0009] It is an object of the present invention to provide an improved contrast agent composition. Another object of the invention to provide an improved uses of the contrast agent composition and an improved method of medical imaging. Moreover, the invention aims at providing improved methods of manufacturing a contrast agent composition.

[0010] It is an achievable advantage if the present invention that the contrast agent composition is metal-free, safe, and completely eliminated from the human body after the MRI exam. It is another achievable advantage of the present invention that the contrast agent composition hyperpolarised state lifetimes that are sufficiently long to enable diagnostic contrast- enhanced MRI.

[0011] Solution according to the invention

[0012] In the following, any reference to one (including the articles “a” and “the”), two or another number of objects is, provided nothing else is expressly mentioned, meant to be understood as not excluding the presence of further such objects in the invention. The reference numerals in the patent claims are not meant to be limiting but merely serve to improve readability of the claims.

[0013] According to one aspect of the invention, the problem is solved by a contrast agent composition with the features of claim 1. The contrast agent composition comprises outer shells, each outer shell enclosing an outer filling. The outer filling comprises a dispersion medium with one or more inner shell(s) dispersed in the dispersion medium. The inner shells(s) each enclose an inner filling that comprises at least one contrast agent. The inner filling has one property and the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic. At least one of the outer shell and the inner shell is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively.

[0014] In the context of the present invention, the term “contrast agent” refers to a substance or composition of substances the presence or absence of which in a location of an object or an object portion that is imaged by means of an imaging method is detectable in the imaging method. An “imaging method” is a method in which a two- or three-dimensional image of the object or the portion of the object is produced. Imaging methods include but are not limited to nuclear magnetic resonance imaging (MRI), x-ray imaging and ultrasound imaging, also referred to as sonography.

[0015] The inner filling may consist of the contrast agent(s) or it may be a mixture of the contrast agent(s) with one or more other materials, for example a solution and / or a suspension of the contrast agent(s) in a solvent or a dispersion medium.

[0016] In the context of the present invention, a shell being “formed by” a surfactant means that the shell is held together by the surfactant molecules, for example by means of the surfactant molecules interacting with each other, with the shell’s filling and / or with a medium in which the shell is dispersed. This does not exclude that the shell moreover comprises other components, such as a polymer. Moreover, the surfactant molecules may comprise a polymer.

[0017] The term “outer surfactant” denotes the surfactant of the inner shell. Likewise, the term “inner surfactant” denote the surfactant of the inner shell. The inner surfactant can be a single surfactant or a mixture of different surfactants. Likewise, the outer surfactant can be a single surfactant or a mixture of different surfactants.

[0018] The invention also encompasses embodiments in which the contrast agent composition in addition to the outer shells that comprise at least one inner shell, there are one or more outer shells without any inner shell. Such outer shells without inner shells may occur if the manufacturing method is unable to ensure that each outer shell comprises at least one inner shell or if ensuring this is unnecessary. Similarly, the invention also encompasses embodiments in which some one or more of the inner shells lack one or more of the contrast agent(s). Such inner shells without contrast agent may occur if the manufacturing method is unable to ensure that each inner shell comprises contrast agent or if ensuring this is unnecessary.

[0019] According to another aspect of the invention, the problem is solved by a use of the contrast agent composition with the features of claim 15. The contrast agent composition is used in a method of nuclear magnetic resonance imaging.

[0020] “Nuclear magnetic resonance imaging” (MRI) is an imaging method that relies on atomic nuclei absorbing radio frequency energy when placed in an external magnetic field, and the nuclei’s resultant evolving spin polarization inducing a RF signal in a radio frequency coil for detection. MRI produces two-or three-dimensional images that indicate the special distribution of the nuclei. Objects imaged by MRI can be non-biological matter, for example samples of materials and apparatus and portions of apparatus, and biological matter, such as biological tissue, biological organisms and parts of biological organisms. In the context of the present invention, the term MRI must not be construed to be limiting to any particular application. In particular, it includes both medical applications and non-medical applications, such as in materials testing.

[0021] According to yet another aspect of the invention, the problem is solved by a use of the contrast agent composition with the features of claim 16. The contrast agent composition is used in a method of medical imaging.

[0022] In the context of the present invention, a “method of medical imaging” is an imaging method in which the object that is imaged is a human or animal body or a portion of a human or animal body. As will be discussed below in further detail, the invention is particularly suitable for medical angiographic and perfusion MRI.

[0023] According to yet another aspect of the invention, the problem is solved by a method of medical imaging with the features of claim 17. The contrast agent composition is contrast agent composition is administered to a patient. The medical imaging method may for example be MRI. In the context of the present invention, the term “patient” includes both human and animal patients.

[0024] According to yet another aspect of the invention, the problem is solved by a method of manufacturing a contrast agent composition with the features of claim 18. The method comprising the steps of: providing an inner filling that has one property of the group of properties comprising lipophilic or lipophobic, which inner filling comprises a contrast agent; providing inner shells with the inner filling so that each inner shell encloses its inner filling; providing an outer filling that is a suspension of the inner shells in a dispersion medium, wherein the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic; and providing outer shells with the outer filling so that each outer shell encloses its outer filling. At least one of the outer shell and the inner shell is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively.

[0025] According to yet another aspect of the invention, the problem is solved by a method of manufacturing a contrast agent composition with the features of claim 19. The method comprises the steps of: providing an inner filling that is lipophobic and comprises a contrast agent; providing inner shells with the inner filling so that each inner shell encloses its inner filling; providing an outer filling that is a suspension of the inner shells in a dispersion medium, wherein the dispersion medium is lipophobic; and providing outer shells with the outer filling so that each outer shell encloses its outer filling. The dispersion medium comprises squalene, squalane or a mixture of squalene and squalane.

[0026] Squalene is a polyunsaturated hydrocarbon, present in shark liver oil. Squalane is a saturated derivative of squalene and well established as established emollient and moisturizing agent. Both oils, squalene and squalane, are in liquid phase at body temperature (36°C), but with different viscosity (12 cP and 31 cP, respectively). Due to this difference in viscosity, it can be achieved with this aspect of the invention that by choosing between squalene and squalene or by choosing a mixture of the two oils at a pre-defined ration, the viscosity of the outer filling can be set to a desired value. Moreover, it is achievable advantage of this aspect of the invention with squalene being part of the dispersion medium that it can protect the inner filling against singlet oxygen induced oxidation. This exploits the fact that squalene is an efficient sacrificial singlet oxygen scavenger. According to a further aspect of the invention, the problem is solved by a method of manufacturing a contrast agent composition with the features of claim 20. The method comprising the steps of: providing an inner filling that has one property of the group of properties comprising lipophilic or lipophobic, and which inner filling comprises a contrast agent; providing inner shells with the inner filling so that each inner shell encloses its inner filling; providing and outer filling that is a suspension of the inner shell(s) in a dispersion medium, wherein the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic; and providing outer shells with the outer filling so that each outer shell encloses its outer filling. The step of providing the outer shells with the outer filling employs electrospraying.

[0027] “Electrospraying” is method that generates fine charged droplets by charging the liquid flowing through a nozzle with an applied electric field. Suitable electrospraying methods are disclosed in He et al, “Structured Mircoe / Nano Materials Synthesized via Electrospray: a Review”, Biometerials Science, 2020, 8, pages 5555-73.

[0028] With the invention, it is achievable that no contrast agent, including water or hyperpolarised protons can leak from the inner filling towards the surrounding of the outer shell or vice versa.

[0029] Preferred embodiments of the invention

[0030] Preferred features of the invention which may be applied alone or in combination are discussed in the following and in the dependent claims.

[0031] A preferred contrast agent composition comprises outer shells, each outer shell enclosing an outer filling. Moreover, A preferred contrast agent composition comprises inner shells each enclose an inner filling. Preferably, at least one of the outer shell and the inner shell of the contrast agent composition is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively. It is an achievable advantage of a surfactant shell that is flexible in the sense that it is reversibly deformable. This can be of particular benefit in applications in which the contrast agent composition is forced to flow within the blood stream through a network of microscopic vessels.

[0032] A preferred surfactant of the inner and / or the outer shell comprises or consists of one or more surfactant(s) that has a relatively large molecular mass. It is an achievable advantage of a large molecular mass that it can contribute to preventing the surfactant from leaving the shell to enter a medium in which the outer shells are suspended. A molecule of a preferred surfactant has a molecular mass greater than 1 kilodalton (kD), preferably greater than 2 kD, more preferably greater than 5 kD, more preferably greater than 10 kD. A preferred surfactant molecule has a molecular mass of less than 60 kD, preferably less than 50 kD, more preferably less than 40 kD.

[0033] A preferred surfactant of the inner and / or the outer shell comprises or consists of one or more surfactant(s) that tend to be hydrophobic. It is an achievable advantage of such hydrophobicity that it can contribute to preventing the surfactant from leaving the shell to enter an aqueous medium in which the outer shells are suspended or an aqueous outer filling. A preferred surfactant has a hydrophilic-hydrophobic balance value of 11 or less, preferably 10 or less, more preferably 9 or less. A preferred surfactant has a hydrophilic- hydrophobic balance value of 3 or more, preferably 4 or more, more preferably 5 or more.

[0034] It is another achievable advantage of a surfactant shell that numerous biocompatible surfactants are available to choose from. Preferably, the outer surfactant and / or the inner surfactant comprises one or more surfactants selected from the list comprising Lecithin-9, non-ionic surfactants based on PEG oligomers with linear or branched alkyl chains (for example like Brij surfactants), PEG oligomers with aryl-alkyl chains (for example Triton X- 100, X-45.X114, X450, Igepal 630, Igepal 720), PEG-20 almond glycerides, PEG-30 dipolyhydroxystearate, PEG-40 sorbitan peroleate, PEG-7 glyceryl cocoate, polysorbate (tween) 61 , polysorbate (tween) 81, sorbitan laurate, sorbitan stearate, sucrose cocoate, glyceryl laurate, ethylene oxide-propylene oxide block copolymers (for example genapol PF 20, genapol PF 30, genapol PF 40, pluronic P85, pluronic L31 pluronic L62) and polyvinyl acetal. Suitable surfactants also include nonionic surfactants including but not limited to alkyl primary, secondary, and tertiary amines, alkanolamides, ethoxylated fatty alcohols, alkyl phenol polyethoxylates, fatty acid esters, glycerol esters, glycol esters, polyethers, alkyl polygycosides, and amineoxides. A preferred surfactant molecule of the inner and / or outer surfactant is a polymer or comprises at least one portion that is a polymer. It is an achievable advantage of a surfactant molecule with a polymer that the polymer can add to the molecular weight of the surfactant molecule. It is another achievable advantage of the polymer that it can add either to the hydrophobicity of the surfactant molecule or to the hydrophilicity of the surfactant molecule (the latter for example in the case of the polymer being a ethylene oxide block copolymers), depending on whether more hydrophobicity or hydrophilicity is desired. Yet, preferably, the polymers or polymer portion of a surfactant molecule is not cross-linked with the polymer or polymer portion of another surfactant molecule. More preferably, the surfactant molecules are not cross-linked with any other molecules, surfactants or not. It is an achievable advantage of the present invention that a hydrophobic part of the surfactant molecule can cover, interact or even interpose to a hydrophobic surface of a dispersed nano- or microobject (capsule or particle) such as the inner shells or the contrast agent.

[0035] The preferred polyvinyl acetal molecule has the structure

[0036] (Formula I).

[0037] It is an achievable advantage of this structure that by varying m, n, p, R1 and R2, the surfactant molecule can adapted to the particular requirements of the intended use of the contrast agent composition. In particular:

[0038] By varying n, the amount of aromatic rings carrying the Ri and R2 residues (these aromatic rings are in the following referred to as “aromatic cores”) can be varied. It is an achievable advantage of this that the hydrophobicity and / or the molecular weight of the surfactant molecule can be adjusted. Moreover, by exploiting the TT-TT interaction (also referred to as stacking) between the aromatic cores, the three-dimensional shape of this part of the surfactant molecule can be adjusted. The n monomers carrying the aromatic cores preferably are hydrophobic. Accordingly, the portion of the surfactant molecule formed of these monomers constitutes a hydrophobic part of the surfactant molecule. The value of n preferably is greater than 10 more preferably greater than 30 even more preferably greater than 50. The value of n preferably is less than 80 more preferably less than 70, even more preferably less than 60.

[0039] Residues Ri and R2 are acryl or substituted acryl groups. It is an achievable advantage of varying R1 and R2 that the hydrophobicity and / or the molecular weight of the surfactant molecule can be adjusted. A preferred residue R1 and / or R2 is a saturated and / or unsaturated fatty acid group. It is an achievable advantage of this embodiment of the invention that the fatty acid groups can mimic phosphatidylcoline (as it is commonly present in natural cell membranes) with regard to its elastic properties. In this regard, the invention can exploit that the residues are in close proximity to each other in the surfactant molecule and to those of other surfactant molecules as the surfactant molecules for the shell. A preferred saturated fatty acid group has 4 or more, preferably 6 or more, more preferably 10 or more C atoms. Preferred saturated fatty acid groups have 20 or less, preferably 18 or less, more preferably 16 or less C atoms. A preferred unsaturated fatty acid group has 1 or more, preferably 2 or more, more preferably 3 or more C atoms. A preferred unsaturated fatty acid group has 10 or less, preferably 8 or less, more preferably 6 or less C atoms.

[0040] By varying p, the amount of vinyl alcohol groups can be varied. It is an achievable advantage of this that the hydrophilicity and / or the molecular weight of the surfactant molecule can be adjusted. The p monomers carrying the aromatic cores preferably are hydrophilic. Accordingly, the portion of the surfactant molecule formed of these monomers constitutes a hydrophilic part of the surfactant molecule. The value of p preferably is greater than 150 more preferably greater than 160 even more preferably greater than 170. The value of p preferably is less than 250 more preferably less than 220, even more preferably less than 190.

[0041] The number m of acetate groups is determined by the hydrolysis conditions in the process of obtaining the starting polymer. Advantageously, by varying m the amphiphilic properties and / or the molecular weight of the surfactant molecule can be adjusted. The value of m preferably is greater than 10 more preferably greater than 20 even more preferably greater than 30. The value of m preferably is less than 100 more preferably less than 90, even more preferably less than 80. In a preferred embodiment of the invention, that the outer shell is formed by the layer of the outer surfactant arranged at the outer surface of the outer filling. It is an achievable advantage of this embodiment of the invention that the outer shell can be provided with biointerface-like elasticity, i.e. an elasticity similar to that of a natural cell membrane, for example that of a blood cell. Particularly advantageously, the outer shell can be reversibly deformable, ie, to deform in response to an external force and elastically recover its initial shape once the external force is removed. Such properties can be of particular advantage when the contrast agent composition is applied for angiographic and perfusion measurements. Advantageously, it is achievable that when the contrast agent composition of the invention is released into the blood stream of a patient, it can adapt to the dynamic in vivo conditions - such as the diverse pressures exerted by the blood flow in the capillaries - in a way similar to that of natural blood cells.

[0042] The present invention also encompasses embodiments in which the inner shells or both the outer shell and the outer shells are formed by the layer of a surfactant arranged at the outer surface of the filling of the respective shell. The inner surfactant and the outer surfactant may be identical, or the surfactant of the inner shell may be a surfactant or surfactant mixture different from the surfactant or surfactant mixture of the surfactant of the outer shell.

[0043] Yet, preferably, the inner shells are formed by a cross-linked polymer or composition of polymers. In the context of the present invention, a shell being “formed by” a cross-linked polymer or composition of polymers means that the shell is held together by the cross-linked polymer. This does not exclude that the shell moreover comprises one or more other components, for example a surfactant. Such surfactant may assist in suspending the inner shells in the dispersion medium of the outer filling.

[0044] It is preferable that all inner shells comprise the same polymer or polymer composition. The preferred polymer of the inner shells is polyurethane (Pll). It is an advantage of Pll that it can produce shells with a good spherical morphology as the inventors have confirmed by means of electron microscopy. Other suitable polymers for the inner shell comprise organic and inorganic polymers, able to form capsules shell in the inverse mini-emulsification process, for example polyurea, polysilicic acid. The invention also encompasses embodiments in which the outer shell is formed by a crosslinked polymer or composition of polymers. The preferred polymer is poly(methyl methacrylate) (PMMA). Other preferred polymers for the outer shell comprise organosoluble polymers, which are soluble in volatile solvents of moderate polarity like ethylacetate, chloroform, THF, but insoluble in superhydrophobic organic solvents, like cyclohexane, hexadecane or paraffin oil, and insoluble in water and low alcohols (methanol, ethanol etc.). Examples include: Polystyrene, Poly(alkylstyrenes) (poly(methylstyrene), poly(butylstyrene etc), polylactides, polyethylacrylate, polybutylacrylate or other polyacrylates and polymethacrylates, Polyvinyl alkanoates (bytyrate, propionate, acetate and their copolymers), polyvinyl acetal, polyvinyl butyral and similar polymers, polyacrylonitrile, polybutadiene, polyisoprene and their copolymers (for example ABS).

[0045] The invention includes embodiments in which both the inner and outer shell are of formed by cross-linked polymer. The inner and the outer can be the same polymer or polymer composition; yet, preferably, they are of different polymer compositions, the composition of each type of shell being chosen to meet this type of cell’s specific requirements.

[0046] The outer filling preferably comprises a dispersion medium with one or more inner shell(s) dispersed in the dispersion medium. The preferred dispersion medium of the outer filling is hydrophobic. Preferably, the dispersion medium comprises or consists of squalene, squalane or a mixture of squalene and squalane. Due to the difference in viscosity of squalene squalane, it can be achieved that by choosing between squalene and squalene or by choosing a mixture of the two oils at a pre-defined ration, the viscosity of the outer filling can be set to a desired value. Advantageously, this can contribute to the outer shell being provided with biointerface- 1 ike elasticity, i.e. an elasticity similar to that of a natural cell membrane, for example that of a blood cell. It is achievable that when the contrast agent composition of the invention is released into the blood stream of a patient, it can adapt to the dynamic in vivo conditions - such as the diverse pressures exerted by the blood flow in the capillaries - in a way similar to that of natural blood cells.

[0047] Preferably, the dispersion medium comprises squalene. It is achievable advantage of this embodiment of the invention that the squalene can protect the inner filling against singlet oxygen induced oxidation. This exploits the fact that squalene is an efficient sacrificial singlet oxygen scavenger. The preferred contrast agent is a nuclear magnetic resonance imaging contrast agent. In the context of the present invention, the term “nuclear magnetic resonance contrast imaging agent” refers to a substance or composition of substances the presence or absence of which in a location of the object or object portion that is imaged by means of nuclear magnetic resonance imaging is detectable by the nuclear magnetic resonance imaging method.

[0048] In the preferred contrast agent composition, the inner filling has one property and the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic. Preferably, the inner filling is hydrophilic. More preferably, the inner filling comprises or consists of water (H2O), heavy water (D2O), or a mixture of water and heavy water. It is achievable advantage of water that it can serve as an MRI contrast agent. Likewise, deuterium advantageously can serve as an MRI contrast agent. Moreover, in a mixture of water and heavy water, the presence of heavy water can affect the spin-lattice relaxation time (T1) of water.

[0049] Moreover, the water, heavy water of mixture of water and heavy water can serve as a solvent or be part of a solvent or as a dispersion medium or part of a dispersion medium of one or more contrast agent. The preferred contrast agent is a MRI contrast agent. If the inner filling comprises multiple contrast agents, the invention also comprises embodiments in which one or more contrast agents a dissolved and one or more others contrast agent(s) are suspended.

[0050] Preferred contrast agents include organic amides and urethanes such as15N-Urea,15N-biurete,15N-ammonim salts of organic and inorganic acids (like15N-ammonium chloride), all water soluble of15N-aminoacids and their salts, all13C-labelled organic water-soluble salts, acids, amides, amines, alcohols like13C-Pyruvate,13C-Urea,13C L-Aspartic acid,13C Sodium formate and13C Sodium acetate.

[0051] The preferred contrast agent is hyperpolarised. This means that nuclei of atoms of the contrast agents - for example hydrogen atoms, deuterium atoms, nitrogen atoms and carbon atoms, in particular,13C atoms - are hyperpolarised. In the context of the present invention, “hyperpolarisation” of a nucleus refers to the nuclear spin being polarised by at least a factor of 10 above the thermal equilibrium condition, preferably by a factor of at least 102, more preferably at least 103, more preferably by a factor of at least 104and most preferably by a factor of at least 105above thermal equilibrium condition. Due to their hyperpolarisation, hyperpolarised nuclei can easily be detected in nuclear magnetic resonance, in particular in MRI imaging.

[0052] In a preferred contrast medium composition, the outer shells are suspended in an aqueous medium, for example water or a physiological saline solution. Suitable physiological slaine solutions are disclosed in Vilvanathan, “Physiological Salt Solutions” in: Lakshmanan et al (eds) “Introduction to Basics of Pharmacology and Toxicology” Springer, Singapore. https: / / doi.org / 10.1007 / 978-981-19-5343-9_11, the relevant portion of which are herewith incorporated into the present disclosure by reference.

[0053] In a preferred use of the contrast agent composition in nuclear magnetic resonance imaging, the contrast medium composition is injected into a blood vessel of the patient.

[0054] Advantageously, thereby an angiographic or perfusion diagnostic procedures can be performed.

[0055] In a preferred method of manufacturing a contrast agent composition, the step of providing the outer shells with the outer filling employs electrospraying. Particular preferably, it employs concentric electrospraying, wherein the emitter comprises an inner capillary and an outer capillary. Preferably, the inner capillary delivers the inner filling, i.e, a suspension of the inner shells, filled with the inner filling and suspended in the dispersion medium. The outer capillary delivers a material of the outer shell or a precursor of this material, preferably in the form of a solution or a suspension of this material. For example, the outer capillary delivers the polymer or polymer composition of the outer shell dissolved in an organic solvent. In the microspraying process, outer shells are formed that enclose the outer filling.

[0056] Brief description of the drawings

[0057] In the following, further preferred embodiments of the invention are illustrated by means of examples. The invention is not limited to these examples, however.

[0058] The drawings schematically show:

[0059] Figure 1 Two cartoon representations (1a and 1b) of the shell structure of Example 4; Figure 2 A scanning electron micrograph (SEM) of of the structure obtained by the electrospraying method of Example 4;

[0060] Figure 3 Confocal laser scanning micrographs (CLSM, 3a and 3b) of the structure of Figure 2;

[0061] Figure 4 A series of CLSM tomography images of the structure obtained by the method of Example 5;

[0062] Figure 5 Further CLSM micrographs (5a and 5b) of the structure of Figure 4;

[0063] Figure 6 A synthesis route for the preparation of a benzaldehyde according to Example 6;

[0064] Figure 7 A synthesis strategy for the preparation of the amphiphilic polyvinyl acetal according to Example 6; and

[0065] Figure 8 Sketch of the Modified microfluidics method and apparatus of Example 7.

[0066] Detailed description of an embodiment of the invention

[0067] In the following description of preferred embodiments of the invention, identical reference numerals refer to identical or similar components.

[0068] Example 1 : Preparing an inner filling

[0069] The inner filling is, for example, deionized water (H2O), heavy water (99,96% D2O,

[0070] CAS 7789-20-0, obtained from Deutero GmbH, Germany, product number number 00507- 10ml-s) or a 1:1 mixture of deionised water and heavy water. In a fourth example, the inner filling is a solution of 100 mg of the hydrophilic contrast agent 15N-Urea per 1 g (gram) of heavy water (D2O). For the encapsulation of water and water-soluble contrast agent any commercially available or synthetic surfactant, having HLB 0-10 and soluble in hydrocarbons (aliphatic, aromatic and / or their mixtures) is suitable. Representative examples are Lubrizol (polyisobutylenesuccinimide pentamine) and PGPR (Polyglyceryl-3 Polyricinoleate).

[0071] Example 2: Encapsulation of the inner filling in inner shells

[0072] 1 g of the inner filling of Example 1 is mixed with 130 mg of a previously prepared homogenized 12:1 mixture of dipropylene glycol and glycerol and stirred for two minutes. Then a previously prepared solution of 50 mg Lubrizol II (polyisobutylenesuccinimide pentamine) in 9 g cyclohexane is added, and the resulting two-phase system is preemulsified by stirring at 700 rpm for 10 min. This pre-emulsion is ultra-sonicated for 120 s (seconds) at 50 % power, pulse 10 s, pause 10 s, with a Branson W450 Digital sonifier under ice-water cooling to obtain a mini-emulsion. Toluene diisocyanate (TDI, 0.215 mL) dissolved in 1 g of cyclohexane is added drop-wisely to the mini-emulsion within 5 min at room temperature, and the reaction mixture is stirred at 700 rpm for 24 h at 25 °C to prompt a polyaddition reaction at the interface of the mini-emulsion’s mini-droplets of inner filling. The resulting Pll shells have sizes of typically between 100 nm (nanometers) and 1000 nm, the average size and the size range depending on the experimental conditions. They Pll wall is rather thin, and they are filled with the inner filling. The shells form stable suspension in cyclohexane, containing some amount of the surfactant Lubrizol U. The suspension can be stored for a long time (up to 6 months were tested) without significant changes of the shape and composition of the capsules, as has been confirmed by dynamic light scattering (DLS), transfer electron microscopy (TEM) and nuclear magnetic resonance (NMR) measurements.

[0073] Example 3: Preparing an outer filling

[0074] The outer filling is prepared by transferring the filled PU shells into a high boiling hydrophobic liquid (with natural or petrochemical origin) dispersion medium such as hexadecane, paraffin oil, squalene or a mixture, for example a 1 :1 mixture, of squalene and squalane. For this, the PU capsules of Example 2 are separated from the cyclohexane by centrifugation and then re-suspended in the dispersion medium by shaking the mixture of PU shells and dispersion medium with Vortex or ultrasonic bath. Example 4: Encapsulation of outer filling in outer shells by means of electrospraying

[0075] Concentric type emitter electrospinning is used to encapsulate the inner filling in outer shells. Details of the electrospraying method can be found in He et al, “Structured Mircoe / Nano Materials Synthesized via Electrospray: a Review”, Biometerials Science, 2020, 8, pages 5555-73, the relevant part of which are incorporated into the present disclosure by reference. The inner needle of the emitter carries the suspension of Pll capsules in oil whereas the outer needle carries a polymer dissolved an organic solvent with relative low boiling point.

[0076] In one example, the polymer is poly(methyl methacrylate) (PMMA). The electrospraying is performed with a commercial platform (Electrospin Cabin V2.1 Dual Voltage, P10003, IME- Technologies) equipped with a positive electrode applied to the spinneret and a counter electrode covered with aluminium foil. The flow of the outer solution and inner dispersion were controlled using two syringe pumps (Aladdin SyringeONE Programmable Syringe Pump, AL-300). The outer needle of the spinning coaxial needle of stainless steel (obtained from SKE Research Equipment) has needle diameters of 0.457 mm 10.254 mm (outer / inner diameter), and the outer needle has needle diameters of 1.83 mm / 1.37 mm (outer / inner diameters). Disposable 3 mL (millilitre) plastic syringes are connected with the coaxial needle via PTFE tubing and Luer Lock connectors.

[0077] The syringe for the outer needle is filled with a PMMA (35 kDa) solution in ethyl acetate (18 wt %, may vary between 15 and 22%). The syringe of the inner needle is filled with 6.2 wt % Pll capsules in paraffin oil with 0.5 % residual Lubrizol II. At a temperature of about 23 °C, good results are obtained using a voltage of between 15 kV (kilovolt) and 20 kV, for example 18 kV, and a tip-collector distance of 17 cm. A suitable pump speed is 0.4 ml / h (millilitres per hour) for the PMMA solution and 0.2 ml / h for outer filling. Scanning Electron microscopy reveals that the PMMA shells are have in diameters of between about 5 pm 20 pm and containing the inner filling.

[0078] Figures 1a and 1 b are two cartoon representations of the resulting shell structure. The inner filling is represented by light blue and dark blue dots. The inner shell is represented in black. Yellow represents the dispersion medium of the outer filling. The PMMA shell is sown in red. In Figures 1a and 1b, the outer shells are suspended in an aqueous suspension medium indicated in light blue. Figure 2 is a scanning electron micrograph (SEM) of the result of the electrospraying method. This outer shell is broken, presumably during the imaging, and the small Pll inner shells are visible inside the outer shell. Figure 3a is a Confocal Laser Scanning Micrograph (CLSM) in reflection mode of the same shell structure, only that this time the inner filling also contains a soluble dye (SR101, sulforhodamine) that fluoresces only in aquatic environment. Figure 3b is such michrograph taken in reflection mode.

[0079] Example 5: Encapsulation of outer filling in GenapolPF 40 surfactant shell

[0080] In this example, the outer shell is formed by a surfactant with relatively low HLB, ranging between 4 and 10. Exemplary surfactants include

[0081] Surfactant Mass (Da) HLB

[0082] Lecithin-9 758 9

[0083] PEG-20 Almond Glycerides mixture 10

[0084] PEG-30 Dipolyhydroxystearate -4000 6

[0085] PEG-40 Sorbitan Peroleate -2000 9

[0086] PEG-7 Glyceryl Cocoate 600 10

[0087] Polysorbate (tween) 61 606 10

[0088] Polysorbate (tween) 81 -4000 10

[0089] Sorbitan Laurate 346 9

[0090] Sorbitan Stearate 430 6

[0091] Sucrose Cocoate mixture 6

[0092] Glyceryl Laurate 639 5

[0093] Genapol PF 20 2500 4

[0094] Genapol PF 30 2600 6

[0095] Genapol PF 40 2800 8

[0096] Pluronic P85 4600 10

[0097] Pluronic L31 5000 5

[0098] Pluronic L62 2500 7

[0099] A outer filling of Pll shells prepared as set out in Example 2 is suspended in a CD3OD (>99,8%, CAS 811-98-3, obtained from Sigma Aldrich, Germany, product number 151947) as set out in Example 3. 6 % wt. of GenapolPF40 (obtained from Clariant Produkte Deutschland GmbH, Germany), dissolved in water and the co-solvent DMF, is added to the outer filling under continuous stirring. In the case of the surfactant GenapolPF40, which poorly dissolvable in water, the cosolvent DMF assists in dissolving the surfactant. The co-solvent is then evaporated at a pressure of 70mbar to form the outer shells.

[0100] Figure 4 is a series of CLSM tomography images of the same inner filling as in figures 3a and 3b but with a GenapolPF40 surfactant outer shell according to the present Example. The images are taken in fluorescence mode at different depth. Figure 5a is a CLSM image of the same structure in transmission mode, and Figure 5b in fluorescence mode.

[0101] Example 6: Preparation of a polyvinyl acetal surfactant

[0102] A Polyvinyl acetal (PVAc) surfactant is manufactured from the nontoxic, biocompatible and biodegradable precursor polymer polyvinyl alcohol (PVA). Aromatic aldehydes doped with hydrophobic functionalities are used for the modification of polyvinyl alcohols in order to obtain a non-ionic polymer with controlled amphiphilic properties. The synthesis strategy for the preparation of the amphiphilic polyvinyl acetal containing hydrophobic aromatic cores is shown in Figure 7. The PVA is combined with an aromatic aldehyde to obtain the PVAc.

[0103] An exemplary aromatic aldehyde is an unsymmetrical benzaldehyde. Figure 6 shows a synthesis route for the preparation of a benzaldehyde containing saturated and unsaturated fatty acid groups in side chains. 3-hydroxy-5-methoxybenzaldehyde or 4-hydroxy-3- methoxybenzaldehyde is selectively acylated with saturated acid chloride; after deprotection of the methoxy group, an unsaturated acid residue is inserted.

[0104] Example 7: Encapsulation of outer filling in PVAc surfactant shell

[0105] In this example, the microfluidic apparatus obtained from Dolomite microfluidics Inc is used to form the outer shells by means of a modified microfluidics method. The apparatus is shown in Figure 8. The method exploits a spontaneous condensation of the surfactant on the surface of droplets of the outer filling, initiated by the gradual decrease of the concentration of the co-solvent (DMSO). A PVAc amphiphilic surfactant is prepared as set out in Example 6. It is then dissolved in water and a suitable co-solvent, in this example DMSO. The role of the co-solvent is to increase solubility of the amphiphilic surfactant, and to lower its viscosity for the purpose of delivering it to a microfluidic chip of the microfluidic apparatus via pump 1. An outer filling with the Pll inner shells suspended in a 1 :1 mixture of squalene and squalane is prepared as set out in Example 3. This outer filling is delivered to Dolomite microfluidics microfluidic chip with pump 2.

[0106] The solution delivered by pump 1 and the suspension delivered by pump 2 are combined in the microfluidic chip, which delivers droplets of the combined fluids to the bottom a beaker (also referred to as a receiver) filled with water. Pump 3 delivers additional water to the beaker in order to keep the relative concentrations of DMSO and H2O in the beaker nearly constant during the process. The water in the beaker is slowly stirred in order to generate a laminar flow that effects the newly generated droplets to avoid mechanical contact to each other.

[0107] In beaker, each droplet slowly ascends, since its density is much lower than the density of the surrounding water. Simultaneously, the co-solvent DMSO diffuses rapidly out of the droplet into the bulk of the beaker. This forces the PVAc molecules to condensate on the hydrophobic surface of the droplet.

[0108] Example 8: Measuring spin-lattice relaxation times

[0109] In the experiments described in the following, the spin-lattice relaxation times Ti of the hydrogen of contrast agent compositions, in which the outer shells are suspended in a fully deuterated dispersion medium, namely CD3OD, CeDi2 (dodecadeutero-cyclohexan) or mixture of CD3OD and D2O, were measured. The results are consistent with essential no water molecules or hydrogen atoms of the inner filling reaching the dispersion medium in which the outer shells are suspended. This suggests that the hydrophobic dispersion medium of the outer filling forms a barrier which holds back the water molecules and the hydrogen atoms of the inner filling. The measurements were performed on an NMR Bruker Fourier® 80 benchtop spectrometer, using the inversion recovery method. 18 points were measured for each sample. Exponential curves were analyzed by the program (Bruker Dynamic Center software (version 2.8.0.1)) and T 1 value was calculated from the fitted function: f(t) = Io * [1 - a*exp (-t / T1 )]

[0110] The spin-lattice relaxation times for bulk pure water, bulk pure heavy water, and a bulk 50% I 50% mixture of them, are shown in Table 1 . In the case of 99.96 % pure D2O, the relaxation time of T1 = 12.5 originates from the 0.04% H2O impurity.

[0111] Material D2O H2O / D2O H2O composition Pure, 99.96 % 50 % : 50 % Pure, MilliQ

[0112] T1 (s) 12.5 7.7 2.77

[0113] Table 1

[0114] Table 2 compares the spin-lattice relaxation times of H2O and bulk D2O and Genapol PF40 surfactant in tetradeuteromethanol (CD3OD). These measurements were made to estimate the effect of a contamination of the CD3OD solvent with H2O. Such impurities would occur if water from the inner filling were able to cross the lipophobic dispersion medium of the outer filling into the to CD3OD dispersion medium surrounding the outer shells. In agreement with the literature, addition of a small amount of D2O to the CD3OD - continuous phase solvent, leads to moderate increase of the T1 time. 0.2% impurities in 99.8% CD3OD contain both CH3OH and H2O. Genapol PF40 is an ethylene oxide-propylene oxide block copolymer and contains end hydroxyl groups. However, they have difference behaviour in NMR experiments.

[0115] H2O, 1 % wt D2O 1 % wt. Genapol PF406 % wt

[0116] Matenal / n CD3OD in CD3OD in CD3OD composition 99,8% purity 99,8% purity 99,8% purity

[0117] T1 (s) 5.3 5.5 6.2

[0118] Table 2

[0119] The spin-lattice relaxation times in samples that contain encapsulated H2O are shown in Table 3. In the case of H2O enclosed in Pll shells prepared as described in Example 2 and suspended in CeDi2, a spin-lattice relaxation times of 1.56 was measured. This much shorter than that shown in Table 1 for of pure H2O (2.77). Since CeDi2 is an orthogonal solvent relatively to water, it is obvious that no water molecules mass transport across the polymer membrane occurs towards the CeDi2 dispersion medium. Molecules of H2O in bulk and in containers of hundred nanometers size may have different relaxation time due to different conditions.

[0120] In comparison, the two columns on the right side of Table 3 show the spin-lattice relaxation times of the same water-filled PU shells, this time suspended in paraffin oil (supplier - Roth, low viscosity) and the suspension enclosed in PMMA outer shells as described in Example 4. The outer shells are re-suspended in the strongly hydrophilic continuous phase solvent CD3OD or a mixture of CD3OD and D2O. The T1 values of 1.44. and 1.21, respectively, suggest that no water molecules mass transport occurs across the double polymer membrane. Water-filled PU capsules, distributed in CeDi2 give a T1 value of 1.56 s. The same capsules, distributed in paraffin oil and encapsulated again in PMMA shell give T1 values of 1.44 and 1.21 seconds, if we have pure methanol-d4 or pure methanol-d4 with 1% D2O. If we expect reasonable diffusion of H2O into outer deuterated solvent, the T1 value should be close to those, described in Table 2 (5-6 s).

[0121] H20, 0.5% wt H2O, 0.5% wt H2O, 0.5% wt encapsulated in encapsulated in encapsulated in PU shell PU-oil-PMMA shells PU-oil-PMMA shells

[0122] Material composition

[0123] Us} 1.56 1.44 1.21

[0124] Table 3

[0125] Similarly, Table 4 shows the spin-lattice relaxation times of the same suspension of water- filled PU shells suspended in paraffin oil (supplier - Roth, low viscosity), but this time the suspension is enclosed in stabilized with GenapolPF40 microdroplets as described in Example 5. The T1 values of 5.44 for normal NMR tube and 5.21 for Shigemi tube, respectively, suggest that no water molecules mass transport occurs across the triple membrane (PU layer, oil phase and surfactant layer of oil microdroplet. H20, 0.5% wt H2O, 0.5% wt encapsulated in encapsulated in

[0126] PU-oil-GenapolPF40 PU-oil- GenapolPF40

[0127] Material capsule capsule composition

[0128] PU-oil- GenapolPF401 PU-oil- GenapolPF401

[0129] D32O D2O / Shigemi tube

[0130] T1 <s) 5.44 5.21

[0131] Table 4

[0132] The features as described in the above description, claims and figures can be relevant individually or in any combination to realise the various embodiments of the invention.

Claims

Claims1. A contrast agent composition comprising outer shells, each outer shell enclosing an outer filling that comprises a dispersion medium with one or more inner shell(s) dispersed in the dispersion medium, wherein the inner shell(s) each enclose an inner filling that comprises at least one contrast agent, and wherein the inner filling has one property and the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic, characterised in that at least one of the outer shell and the inner shell is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively.

2. The contrast agent composition of claim 1 , characterised in that the outer surfactant and / or the inner surfactant has a hydrophilic-hydrophobic balance value of between 3 and 11.

3. The contrast agent composition of claim 1 or 2, characterised in that the outer surfactant and / or the inner surfactant comprises one or more surfactants selected from the list comprising Lecithin-9, PEG-20 almond glycerides, PEG-30 dipolyhydroxystearate, PEG- 40 sorbitan peroleate, PEG-7 glyceryl cocoate, polysorbate (tween) 61 , polysorbate (tween) 81 , sorbitan laurate, sorbitan stearate, sucrose cocoate, glyceryl laurate, genapol PF 20, genapol PF 30, genapol PF 40, pluronic P85, pluronic L31 pluronic L62 and polyvinyl acetal.

4. The contrast agent composition of claim 3, characterised in that the surfactant of the outer shell and / or the surfactant of the inner shell comprises a polyvinyl acetal on molecule of Formula I(Formula I), where n is between 10 and80, m is between 10 and 100 and is p is between 150 and 250, and wherein each of Ri and R2 is an acryl or a substituted acryl group.

5. The contrast agent composition of any one of claims 1 to 4, characterised in that the outer shell is formed by the layer of an outer surfactant arranged at the outer surface of the outer filling.

6. The contrast agent composition of any one of claims 1 to 5, characterised in that the inner shell(s) are formed by a cross-linked polymer or composition of more than one polymer.

7. The contrast agent composition of claim 6, characterised in that the cross-linked polymer is selected from the group comprising polyurethane, polyurea, and polysilicic acid.

8. The contrast agent composition of any one of claims 1 to 7, characterised in that the dispersion medium of the outer filling is hydrophobic and the inner filling is lipophobic.

9. The contrast agent composition of any one of claims 1 to 8, characterised in that the inner filling comprises water, heavy water or a mixture of water and heavy water.

10. The contrast agent composition of any one of claims 1 to 9, characterised in that the inner filling comprises water, heavy water or a mixture of water and heavy water as a contrast agent.

11. The contrast agent composition of any one of claims 1 to 9, characterised in that the inner filling is an aqueous solution or an aqueous suspension of the one or more contrast agent(s).

12. The contrast agent composition of any one of claims 1 to 11, characterised in that the at least one of the contrast agent(s) is a nuclear magnetic resonance imaging contrast agent.

13. The contrast agent composition of any one of claims 1 to 12, characterised in that the at least one of the contrast agent(s) is hyperpolarised.

14. The contrast agent composition of any one of claims 1 to 13, characterised in that the outer shells are suspended in an aqueous dispersion medium.

15. A use of the contrast agent composition of any one of claims 1 to 14 in a method of nuclear magnetic resonance imaging.

16. A use of the contrast agent composition of any one of claims 1 to 15 in a method of medical imaging.

17. A method of medical imaging in which a contrast agent composition according to any one of claims 1 to 14 is administered to a patient.

18. A method of manufacturing a contrast agent composition the method comprising the steps of:Providing an inner filling that has one property of the group of properties comprising lipophilic or lipophobic, and which inner filling comprises a contrast agent;Providing inner shells with the inner filling so that each inner shell encloses its inner filling;Providing an outer filling that is a suspension of the inner shells in a dispersion medium, wherein the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic; andProviding outer shells with the outer filling so that each outer shell encloses its outer filling; characterised in that at least one of the outer shell and the inner shell is formed by a layer of an outer surfactant arranged at the outer surface of the outer filling or an inner surfactant arranged at the outer surface of the inner filling, respectively.

19. A method of manufacturing a contrast agent composition the method comprising the steps of:Providing an inner filling that is lipophobic and comprises a contrast agent;Providing inner shells with the inner filling so that each inner shell encloses its inner filling;Providing an outer filling that is a suspension of the inner shells in a dispersion medium, wherein the dispersion medium is lipophobic;Providing outer shells with the outer filling so that each outer shell encloses its outer filling; characterised in that the dispersion medium comprises squalene, squalane or a mixture of squalene and squalane.

20. A method of manufacturing a contrast agent composition the method comprising the steps of:Providing an inner filling that has one property of the group of properties comprising lipophilic or lipophobic, and which inner filling comprises a contrast agent;Providing inner shells with the inner filling so that each inner shell encloses its inner filling;Providing and outer filling that is a suspension of the inner shell(s) in a dispersion medium, wherein the dispersion medium has the other property of the group of properties comprising lipophilic or lipophobic; andProviding outer shells with the outer filling so that each outer shell encloses its outer filling; characterised in that the step of providing the outer shells with the outer filling employs electrospraying.

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