Diblock polymer

Abstract

A nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-5 guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4;10 wherein said nanoparticle comprises a chelator such as DOTA; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

Description

[0001] Diblock Polymer

[0002] This invention relates to nanoparticles comprising diblock polymers comprising an oligo- or polyguluronate or oligo- or polygalacturonate component linked to a second polymer component, such as a dextran or polyalkylene glycol. The invention further relates to the uses of the nanoparticles to deliver metal ions, such as radionuclides, to a patient.

[0003] Background of Invention

[0004] Alginates are algal or bacterial polysaccharides much utilised in foods, pharmaceuticals etc. because of their mild and useful gelation properties. Most alginates have high affinities for multivalent cations like Ca ions, the binding of which leads to hydrogel formation. These phenomena are linked to the presence in alginates of sequences (blocks) of L-guluronic acid (G), which co-exist with blocks of D-mannuronic acid (M) and alternating (..MG..) blocks. Figure 1 shows the structure of L-guluronic acid residues present in alginate and shows a theoretical distribution of these units with an alginate chain.

[0005] The content and distribution of G depends on the organism from which the alginate derives and is a result of the action of a family of mannuronan 05 epimerases.

[0006] Alginates may themselves be classified as block polysaccharides, the length and distribution of the three block types varying due to the inherent compositional heterogeneity of alginates. The relationship between the gelling properties of alginates with multivalent cations and the structure, sequence and chain length of alginates has been extensively investigated for decades.

[0007] It is well known how (almost) pure G blocks can be isolated from the parent alginate and separated (a standard method is partial hydrolysis combined with fractional precipitation with dilute acid: G-blocks precipitate selectively when alginate is hydrolysed at a specific pH (M- and MG blocks are soluble). In contrast, the properties of isolated M- and G-blocks and their incorporation in precisely engineered alginate-based block polysaccharides have been minimally investigated.

[0008] The present inventors have now determined that nanoparticles can be prepared from precisely engineered alginate-based block polysaccharides in which G blocks are linked in any convenient fashion to a second polymer such as an oligo or polysaccharide. These polymer conjugates can spontaneous for nanoparticles in the presence of metal ions. The G blocks required are ideally ones that contain a high proportion of G residues as it is these units that coordinate the metal ions and allow the spontaneous formation of nanoparticles in solution.

[0009] The importance of these diblock polymers has been reported in WO2023 / 025943 however the inventors have now appreciated a series of important further developments to this technology.

[0010] Summary of Invention

[0011] Viewed from one aspect therefore, the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises Ra, Ba or Sr ions in combination with other metal ions such as Ca ions.

[0012] Viewed from another aspect the invention provides a process for the preparation of a nanoparticle comprising:

[0013] (I) obtaining a first diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonate residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; (II) combining said first diblock polymer with a second diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4 and wherein said second diblock polymer additionally comprises a chelator or biological targeting molecule or is functionalised to carry a reactive group to which a chelator or biological targeting molecule can be covalently bound after nanoparticle formation; so as to form a mixture; and subsequently:

[0014] (III) contacting said mixture with metal ions to form nanoparticles; and, if required,

[0015] (IV) reacting said nanoparticles with a chelator or biological targeting molecule so as to covalently bind said chelator or biological targeting molecule to said nanoparticles via said reactive group.

[0016] In one embodiment, this process can involve a third diblock polymer as defined herein carrying a further different chelator or the process of step (II) can involve one diblock polymer as defined herein carrying a chelator and one diblock polymer carrying a reactive group capable of reaction after NP formation.

[0017] Viewed from another aspect the invention provides a process for the preparation of nanoparticles comprising:

[0018] (I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4, such as a polysaccharide or polyalkylene glycol;

[0019] (II) combining said diblock polymer with an L-guluronic acid oligomer or galacturonic acid oligomer comprising at least 50 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation n where n is at least 3 so as to form a mixture; and subsequently:

[0020] (III) contacting said mixture with metal ions to form nanoparticles.

[0021] Viewed from one aspect the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an poly / oligogalacturonate comprising at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said diblock polymer forms a nanoparticle spontaneously from an aqueous solution comprising metal ions, e.g. in a concentration of at least 0.1 mM of metal ions.

[0022] Viewed from one aspect the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises a chelator such as DOTA; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

[0023] Viewed from one aspect the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises a biological targeting ligand such as folic acid / folate; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

[0024] Viewed from one aspect the invention provides a nanoparticle which forms spontaneously from an aqueous solution comprising metal ions (e.g. in a concentration of at least 0.1 mM of metal ions); and a blend of a first and second diblock polymer; said first diblock copolymer comprising: a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% guluronic acid residues and having a degree of polymerisation of at least 4; and wherein said second diblock polymer comprises a first component covalently bound via a linker to a second component; wherein said first component is an poly / oligogalacturonate comprising at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% galacturonate acid residues and having a degree of polymerisation of at least 4.

[0025] Metal ions used herein are preferably metal 2+ or 3+ ions.

[0026] Viewed from another aspect the nanoparticles of the invention are core shell nanoparticles wherein said first component forms the core and said second component forming the shell of said nanoparticle, wherein metal ions are ionically bound within the core of the nanoparticle.

[0027] In order to form the nanoparticles, it is particularly preferred if the contact between the diblock polymer and the ions is effected by dialysis or internal gelling, e.g. caused by a slow adjustment of the pH releasing a gelling ion from a suitable salt or ion complex.

[0028] Viewed from another aspect the invention provides use of a nanoparticle as hereinbefore defined to deliver a metal ion or charged organic compound to a patient.

[0029] Detailed Description of Invention

[0030] This invention uses diblock polymers and their ability to form nanoparticles that coordinate a positive metal ion. Such nanoparticles might have important medical applications, e.g. to allow delivery of the positive metal ion to a patient.

[0031] What is surprising is that by terminally attaching a second polymer such as dextran to G-alginate (= oligoguluronate = G-blocks) or poly / oligogalacturonate well-defined, highly stable, nanoparticles can be formed when positive ions such as calcium ions are contacted with the diblock polymer.

[0032] In contrast, alginates themselves (i.e. without the G-block concentration required in the present invention) tend to form hydrogels in the presence of the aqueous metal ion solution and G-blocks alone form precipitates.

[0033] The invention requires the combination of a first block (or first component) which is typically a L-guluronic acid oligomer. Whilst the use of the defined G- blocks (oligoguluronate blocks) is preferred, in one embodiment the oligoguluronate blocks can be wholly or partially replaced with poly / oligogalacturonate blocks. Such blocks can be obtained from pectin.

[0034] The invention requires the combination of a first block (or first component) which is a L-guluronic acid oligomer or poly / oligogalacturonate and a second block (or second component) which is a polymer such as an oligo or polysaccharide or polyalkylene glycol. Ideally the second polymer is water soluble. The term water soluble is used herein to define a material which has a solubility in water of at least 10 g / L at 20°C.

[0035] The second polymer should be attached terminally to the L-guluronic acid oligomer or poly / oligogalacturonate, i.e. via functionality at the end of the L- guluronic acid oligomer or poly / oligogalacturonate. It is also preferred if the second polymer is connected via a terminal position. The diblock can therefore be considered “linear”, i.e. where both blocks are connected via terminal positions on each respective block. Guluronic acid oligomers or poly / oligogalacturonates

[0036] In one embodiment, the invention requires the use of guluronic acid oligomers (G oligomer) or poly / oligogalacturonate as the first component in the diblock polymer. These oligomers are readily obtained from alginate or pectin.

[0037] Native alginate chains do not contain a sufficient concentration of G residues and hence the native alginate should be subjected to hydrolysis, e.g. in acid or base, to generate guluronic acid oligomers in which the content of guluronic acid residues is higher. Guluronic acid oligomers of interest are L-guluronic acid oligomers.

[0038] The alginate from which the guluronic acid oligomers are prepared is preferably one with a high guluronic acid content. Such alginates are known. It may be that different native alginates can be used to generate guluronic acid oligomers of different degrees of polymerisation.

[0039] The use of acid hydrolysis, e.g. using a strong acid such as sulphuric or nitric acid, is preferred as a method for degrading the natural alginate chains. The hydrolysis process can be effected simply by exposing the native alginate to the acid or base. Conveniently this can be effected at room temperature but elevated temperatures can also be used. Stirring of the reaction mixture ensures fractionation occurs efficiently.

[0040] Poly / oligogalacturonate blocks can be obtained from pectin. The poly / oligogalacturonate blocks can be demethylated (either chemically or enzymatically) to provide demethylated poly / oligogalacturonate blocks, such as demethylated poly / oligogalacturonate blocks. Instead therefore of the alginate G- blocks primarily described herein, these poly / oligogalacturonate blocks can be used. Alternatively, diblock polymers might be devised in which a blend of poly / oligogalacturonate blocks and L-guluronate blocks are used. Whilst the invention is generally described with reference to G-blocks, in any scenario, the poly / oligogalacturonate blocks may be substituted for the G-blocks. The poly / oligogalacturonate blocks may be poly / oligo-L-galacturonate blocks or poly / oligo-D-galacturonate blocks.

[0041] Pectins are complex polysaccharides that are abundant in nature because they are present in the cell wall of the majority of terrestrial plants and some species of algae, and they provide rigidity. In addition to neutral sugars (L- rhamnose, D-galactose, L-arabinose, apiosis, etc.), pectins are currently the main known source of D-galacturonic acid. The hydrolysis of pectins, in order to obtain galacturonates in the form of oligomers is known. Numerous studies of chemical, physical or enzymatic treatments have been considered alone or in combination to depolymerize the pectins. Acid hydrolysis (WO 2009 / 004153) allows the deacetylation and hydrolysis of esters as well as the elimination of neutral sugars (L-rhamnose, L-arabinose, D- glucose, D-xylose, D-galactose, etc.) but the glycosidic linkages between galacturonic units are more resistant, and the reactions result in oligogalacturonates.

[0042] Pectinases are a heterogeneous group of enzymes that can hydrolyze the frequent methyl ester functions on galacturonic units, or eliminate neutral sugars from pectins (rhamnogalacturonases, arabinases, galactanases, etc.). Enzymes can also be used to depolymerize the galacturonic chains.

[0043] Shorter galacturonic chains can be obtained by the hydrolysis of the glycosidic bonds of the polygalacturonic acids under the action of the poly (methyl) galacturonases oligogalacturonates.

[0044] Poly / oligogalacturonate blocks or guluronic acid oligomers of use in the invention may have a degree of polymerisation in the range of 3 to 100, such as 5 to 80, especially 10 to 50. A further preferred range is 10 to 40. In practice, it is challenging to obtain very long galacturonate blocks from pectins and hence the use of shorter blocks with a DP of 32 to 50 is preferred.

[0045] The nanoparticles (NPs) of the invention may have many diagnostic and / or therapeutic uses and hence they need to offer stability in physiological conditions. The inventors have surprisingly found that improvements in physiological stability can be achieved if the G-block length is long, i.e. with a degree of polymerisation of at least 20. Guluronic acid or poly / oligogalacturonate oligomers of 20 to 40 repeating units are ideally used. Longer G-blocks also stabilize NPs and prolong nanoparticle lifetime in physiological saline and may therefore be important for storage as the NPs of the invention may need to be stored for periods before use. On the other hand slow but controllable disintegration under physiological conditions may reduce possible accumulation of nanoparticles in e.g. the liver.

[0046] The degree of polymerisation can be determined via NMR and represents the number of all monomer residues within the oligomer. As noted below, not all these monomer residues are guluronates or galacturonates but at least 50% of them must be guluronate / galacturonates residues. The degree of polymerisation in G-blocks can be controlled via the length of the hydrolysis step and by the nature of the native alginate on which the hydrolysis is effected. Longer hydrolysis reaction leads to lower degrees of polymerisation and vice versa. For the avoidance of doubt DP = degree of polymerization = number of monomers per chain. For example, a polymer GGGGG, GGGGM, or MGMGM have a DP = 5 (i.e. n=5).

[0047] The degree of polymerisation of the guluronic acid or oligogalacturonate oligomer is generally chosen depending on the nature of the positive ion being coordinated and on the nature of the second copolymer. If the degree of polymerisation of the guluronic acid or oligogalacturonate oligomer is low then to ensure the formation of nanoparticles, the second polymer tends to have a higher degree of polymerisation (DP). In general, if the metal ion being coordinated binds more strongly to the alginate (e.g. Ba) then lower degrees of polymerisation might be employed than if the metal ion is smaller, e.g. Ca.

[0048] Alternatively viewed, the weight average molecular weight (Mw) of the guluronic acid or oligogalacturonate oligomers may be in the range of 1000 to 40,000. Mw can be determined using GPC, light scattering, or a combination of both.

[0049] It will be appreciated that guluronic acid oligomers may be prepared from alginate by methods known in the art including hydrolysis, enzymic degradation (e.g. using lyases), or alkaline degradation. The skilled person can devise suitable methods for forming these oligomers.

[0050] More accurate control of oligomer chain length and oligomer make-up can be achieved by combining acid hydrolysis with chromatography (SEC). For example, such a technique can be used to control the residual M (D-mannuronic acid) content in the oligomer to 2-12%. Moreover, the residual M units can be located at the chain terminus (non-reducing end).

[0051] Guluronic acid oligomers may contain some other monomer residues however it is essential that the guluronic acid content in the guluronic acid oligomers is at least 50 mol%, preferably at least 70 mol%, especially at least 85 mol%. The idea is to prepare guluronic acid oligomers in which the guluronic acid concentration is much higher than in the native alginate. The alginate is fractionated and oligomers which are lower in guluronic acid are removed. Only the oligomeric blocks with high G content are interesting. High G content improves the metal ion binding selectivity. Alternatively viewed, the guluronic acid oligomer is one in which 50% or more of the monomer residues are L-guluronic, preferably 70 % or more such as 85 % or more of the monomer residues. The FG value therefore is 0.5 or more, such as 0.7 or more, especially 0.85 or more. The use of pure guluronic acid oligomers is, of course, possible (e.g. 99 mol% or more of an FG of 0.99). Other residues that might be present in the guluronic acid oligomers present include mannuronate.

[0052] The hydrolysis reaction leads to break up of the polymer chains and the target guluronic acid oligomers can be fractionated from the mix of oligomers that form.

[0053] It will be appreciated that a mixture of guluronic acid oligomers might be used when preparing the diblock polymers of the invention. Once the native alginate is hydrolysed and the high G content oligomers are isolated, such a mixture might be used as the first component in the diblock polymers of the invention or further purification might be used to isolate a single oligomer or a mixture containing fewer different oligomers. The skilled person can tailor the nature of the guluronic acid oligomer first component depending on the required properties of the nanoparticles. What is required however is that the mixture contains oligomers in which substantially all the components have at least 50 mol% guluronic acid residues.

[0054] In oligogalacturonate blocks, it is also preferred if the content of galacturonate acid residues is at least 50 mol%, preferably at least 70 mol%, especially at least 85 mol%.

[0055] Determining the number of repeating units within the oligomer and determining the number of residues within the oligomer can be achieved using known analytical techniques such as NMR. MALS, SEC-MALS and viscometry can also be used to determine the Mw of a polymer and that information can also be used to determining the number of repeating units or monomers within a polymer.

[0056] It is easier to remove non galacturonates residues than in the G-block case and hence preferably at least 90 wt% of the oligomer is galacturonates.

[0057] The guluronic acid oligomers or galacturonate oligomers must then be linked to the second polymer via any convenient chemistry. The nature of the hydrolysis of the alginate means that the guluronic acid oligomers contain a carbonyl group, such as aldehyde functionality. This carbonyl, or specifically aldehyde, functionality can be exploited when joining the guluronic acid oligomers to the second polymer. This carbonyl functionality is preferably positioned at the end of the guluronic acid oligomer. Chemistry for binding a linker to galacturonates is also well known.

[0058] Linker

[0059] The first component is joined to the second component via a linker. The nature of the linker is not crucial and the skilled chemist can devise many ways of joining a guluronic acid or galacturonate oligomer to a second polymer. In theory this linker could simply be one atom that allows the two components of the diblock polymer to be linked, e.g. an -O- atom. Preferably however a dedicated linking molecule is used.

[0060] Any suitable covalent chemistry might be used with suitable functionalisation of reactants to create appropriate nucleophiles and electrophiles. The use of click chemistry is a particularly preferred method for joining the larger molecules. For example, an aminooxy-azide is readily reacted with an aminooxy-DBCO in a well- known click chemistry reaction. Functionalisation of the reactants with complementary click groups allows simple connection of the reactants. The linker in this embodiment therefore becomes the atoms between the L-guluronic acid oligomer and the second polymer. A preferred linker may therefore include a triazole group (formed by the click reaction of the alkyne and azide).

[0061] The linker of the invention is preferably multifunctional, such as difunctional or trifunctional. In one embodiment, a single linker is used that is difunctional, i.e. it must be capable of reacting with both reactants. The linking of the two components can be effected simultaneously but more conveniently one of the component is first reacted with the linker and subsequently the other component is reacted with the functionalised component.

[0062] Ideally, the linker is a small molecule with an Mw of less than 300 g / mol, such as 50 to 200 g / mol. It is however, possible to use larger linking groups such as a polyalkylene oxide chain. Preferably such a polymeric linker will have fewer than 20 repeating units, such as less than 10 reepating units.

[0063] Conveniently, the linking reaction will exploit terminal masked carbonyl / aldehyde groups in the guluronic acid oligomers and second polymer, if present. Ideally therefore, the linking reaction involves a reductive amination, amination or reaction involving click chemistry, e.g. with a functional group selected from azide, alkyne, thiol, alkene etc. The use of a dioxyamine or a dihydrazide is preferred. The linker may therefore form a Schiff base (oxime or hydrazone) with the first or second components. Conveniently, one of the components is functionalised with a difunctional reductive amination type reagent, such as a O,O''-1 ,3,- propanediylbishydroxylamine dihydrochloride or adipic acid dihydrazide (ADH). The other component is then combined to link the two blocks. Details are provided in the experimental section below and can be readily adapted by the skilled chemist.

[0064] Conveniently, the linker is a difunctional linker in which there are terminal functional groups linked by an alkylene chain, such as a C1-10 linear alkylene chain. Functional groups of interest include O-NH2 or -CO-NH-NH2. Longer linkers might contribute to size of the diblock polymer so linker length is a further tool that the skilled chemist can use to change the properties of the diblock polymer and the nanoparticle containing it.

[0065] Once the reaction is complete, the Schiff bases might be reduced, e.g. to form a stable amine). Suitable reducing agents include picoline borane or sodium cyanoborohydride. Such a species might be chemically more stable than an oxime or hydrazone.

[0066] In Figure 2, there is a description of the reaction of guluronate with PDHA or ADH to form the oxime or hydrazone with subsequent reduction to the N-oxide or hydrazine. It will be appreciated that the hydrazone has an equivalent form - a pyranoside. There might also be equivalent cyclic forms, such as furanosides.

[0067] Ideally, the linker should link terminal positions of the guluronic acid oligomer and the second polymer.

[0068] The skilled person will be readily able to devise suitable chemistry to link the two components. In one embodiment, the linker might contain 5 to 20 backbone atoms (i.e. the chain linking the two blocks is 5 to 20 atoms in length). For example, a O-CH2-CH2-CH2-CH2-O linker contains 6 backbone atoms. Conveniently, the first or second oligomer / polymer is bound to a difunctional linker and subsequently the other required component is bound to the difunctional linker. If required protection chemistry can be used to ensure only functional group of a difunctional linker reacts.

[0069] In some embodiments, the linker may comprise a short chain polyalkylene glycol, such as a PEG. Such a chain may have up to 10 repeating units, e.g. up to 5 such units. The skilled person can use known purification technology to purify the diblock polymer by removing unreacted components, e.g. by dialysis, fractional precipitation, chromatography, sequential extraction.

[0070] Second polymer

[0071] The second component in the diblock polymer is a polymer such as an oligo or polysaccharide, poly(meth)acrylate or polyalkylene glycol. It will be appreciated that the second soluble polymer must be different from the guluronic acid or galacturonic acid oligomer. The second polymer does not therefore contain more than 30 mol% guluronic acid or galacturonic acid residues. Ideally, it does not contain any guluronic acid or galacturonic acid residues. The second polymer is preferably not one that derives from alginate or pectin.

[0072] It is preferred if the second polymer is a water-soluble polymer. Some insoluble polymers may also be used, especially those with a low degree of polymerisation, such as insoluble chitin oligomers with a DP of 6 to 40.

[0073] The second polymer is one that, when linked to the first component, forms a nanoparticle in the presence of positive ions such as metal ions. Second polymers that form a precipitate in those circumstances are excluded.

[0074] It is preferred if the second polymer has a higher weight average molecular weight (Mw) than the first oligomer. Ideally, the second polymer has a Mw at least 2 times that of the first oligomer, such as 3 to 8 times higher. If the second polymer has a Mw which is too high however (e.g. 20x or more the Mw of the first oligomer) then it is more likely that a precipitate forms rather than the target nanoparticle.

[0075] Alternatively viewed, the degree of polymerisation of the second polymer should be the same as or higher than that of the first oligomer.

[0076] In one embodiment, the ratio of n to m is therefore important where n is the DP of the guluronic acid or galacturonate and m is the DP of the second polymer. The ratio is ideally 2:1 (n:m) to 1:9 (n:m), such as 1 :1 (n:m) to 1:9 (n:m), especially 1 :1.5 to 1 :7. A particularly preferred ratio is 4n => m >=n.

[0077] In general therefore, if the DP of G is much larger than the DP of the second polymer then precipitation occurs. If both oligomers are short, e.g. the DP is less than 15 for both oligomers then if the DP of G is the same as the DP of the second polymer then precipitation occurs rather than the formation of NP. If therefore the DP of the G oligomer is in the range of n=3 to 15 then the DP of the second polymer m is preferably 30 to 180. If the value of m exceeds 180 then there is a risk that the diblock polymer is water soluble and hence m is preferably 180 or less.

[0078] The exact values of m and n which lead to precipitation or nanoparticles may vary depending on the nature of the positive ion being coordinated within the nanoparticle.

[0079] Without wishing to be limited by theory, it is believed that the appropriate Mw of DP of the second polymer encourages the spontaneous formation of nanoparticles in an appropriate medium, typically an aqueous medium.

[0080] The Mw of the water-soluble polymer may also be less than the guluronic acid oligomer if both polymers have at least 20 repeating units. It is particularly preferred if the first polymer has a DP of 20 or more such as 20 to 40. G blocks of this length offer improved stability under physiological conditions.

[0081] Determining the number of repeating units within the second polymer can be achieved using well known analytical techniques such as NMR. MALS, SEC- MALS or viscometry can also be used to determine the Mw of a polymer and that information can also be used to determining the number of repeating units (monomers) within a polymer. Many commercial polysaccharides are sold with a specified degree of polymerisation.

[0082] It can be considered in fact that the water-soluble polymer forms a shell where the guluronic acid oligomer forms the core of a core shell nanoparticle. The nanoparticles can be regarded as micelles or polymersomes therefore.

[0083] A preferred water-soluble polymer is polyethylene glycol or an oligo or polysaccharide, especially hyaluronan, pullulan, p-1 ,3-glucan, heparin, glycosaminoglycans, amylose, chitosan or dextran. Dextrans are branched poly-a- D-glucosides of microbial origin having glycosidic bonds predominantly C-1 — > C-6". Dextran chains are of varying lengths.

[0084] The second component is preferably a dextran or polyethylene glycol. If the second component is a polyethylene glycol then it is preferred if the DP is 5 to 200. Where the second component is a PEG, this chain might be interrupted by a linker group to allow a PEG chain on the diblock polymer to bind to a PEG chain on an additional moiety such as a biological targeting molecule or chelator. In that scenario, the DP is the total number of PEG units present. The term biological targeting molecule means a molecule such as an antigen, antibody, protein, aptamer, nucleic acid, vitamin etc that might be used to target a specific site within the body or specific cells such as cancer cells. The nature of the biological targeting molecule is not significant.

[0085] The water-soluble polymer can be functionalised to carry a linker as hereinbefore described and a linking reaction between the first component and water-soluble polymer can then be effected.

[0086] If the second component is a polyalkylene glycol ideally it contains at least 10 repeating units.

[0087] In a highly preferred embodiment, the first component is linked to a dextran, ideally via reductive amination, i.e. the linker comprises an N-oxide or hydrazine.

[0088] It will be appreciated that second polymers of appropriate chain length may be prepared by methods including acid hydrolysis, enzymic degradation, or alkaline degradation, e.g. for second polymers such as hyaluronan or dextran.

[0089] In one embodiment, the invention provides an oligomer comprising at least 50 mol% L-guluronic acid residues and having a degree of polymerisation n where n is at least 3 bound for a linker to hyaluronic acid. Hyaluronic acid is a natural polysaccharide that specifically can recognize receptors overexpressed by tumor cells, enabling enhanced cancer retention.

[0090] Diblock Polymers

[0091] Engineered diblock polymers of the invention therefore comprise, such as consist of, two or more different blocks linked through a suitable conjugation method. Diblock polymers of the invention may be linear.

[0092] Diblock polymers of the invention can be named Gn-L-xxx herein where G is the guluronic or galacturonate oligomer with degree of polymerisation n. L is the linker and xxx is the second polymer, such as dextran. In particular, the diblock polymer is Gn-L-Dexmwhere Dex is dextran and m is the degree of polymerisation of the dextran. In particular, the diblock polymer is Gn-L-PEGmwhere m is the degree of polymerisation of the PEG. As noted above, the PEG may be interrupted by a linker.

[0093] The value of n is preferably 8 to 70. The value of m is preferably 30 to 180, such as 30 to 150. Ideally m is at least 2n.

[0094] The ratio of n to m is also important. The ratio is ideally 2:1 to 1 :9. It is preferred therefore that 9n > m > n / 2. A particularly preferred ratio is 4n => m >=n.

[0095] Nanoparticles of the invention can, of course, be made from a mixture of diblock polymers. In particular, it will be appreciated that the guluronic acid or galacturonic acid may have a polymer length distribution. Also, the second polymer may also have a degree of polymer length distribution, and nanoparticles can hence be made from such diblock polymer mixtures.

[0096] Nanoparticles

[0097] The diblock polymers of the invention self-assemble under defined conditions where one of the blocks can develop short-range attractive interactions while the other ones develop long-range repulsive interactions. Self-assembly is a spontaneous process leading to a great diversity of structures whose characteristics depends on the molecular parameters of the starting block polymers. The diblock polymers are preferably dissolved in water. On the addition of metal ions, nanoparticles form. Without being limited by theory, it is envisaged that the presence of metal ions initially allows the formation of dimers of the diblock polymers. The formation of these dimers leads, in turn to the formation of nanoparticles. The nanoparticles of the invention can consist of the diblock polymer or a mixture of diblock polymers (as well as any metal ion and biological targeting moiety / chelator).

[0098] Normally an excess of metal ions is added to ensure nanoparticle formation. The concentration of metal ions required in solution varies depending on the nature of the metal ion. It will also be appreciated that a mixture of metal ions might be used. In general, the concentration of metal (2+) ions required in solution follows the order: Mg » Mn > Ca > Sr > Ba > Cu > Pb. In some embodiments, a saturated solution might be used.

[0099] The addition of metal ions to an aqueous solution of the diblock polymer allows the spontaneous formation of the nanoparticles of the invention. Ideally, addition of the metal ions occurs using dialysis or internal gelation. It may be convenient to start from a salt form, such as a sodium salt form of the diblock polymer. Addition of metal ions, e.g. calcium ions, in a controlled manner, e.g. via titration, dialysis allows nanoparticles to be formed. Conveniently the diblock polymer is in an aqueous salt form. Metal ions, such as calcium ions can be added to cause the nanoparticle formation from a convenient source such as a complex (e.g. EDTA complex) or metal salt. The salt is preferably water soluble. Other complexes include DOTA, NOTA, TGA, NOTAGA, TETA, PCTA, TRAP, MACROPA++. It is also possible to deliver metal ions from a water insoluble salt through pH manipulation. In one embodiment, calcium ions can be delivered slowly into the aqueous solution of the diblock polymer from calcium sulphate. Calcium sulphate is insoluble but its solubility can be increased by reducing the pH of the solution

[0100] Internal gelation is a process where metal ions such as Ca is first distributed in the alginate, for example as metal carbonate microparticles, or as soluble metal complex, such as metal-EGTA or metal-EDTA complexes. A pH adjuster such as GDL is used to slowly lower pH sufficient to release metal ions from the source to induce metal-alginate gelation. In the presence of the diblocks of the invention this surprisingly gives stable nanoparticles.

[0101] Conveniently, dialysis involves a diblock solution dialysed against a metal ion solution such as a solution of Ca ions, e.g. CaCh . The length of the dialysis can vary depending on the molecular weight of the diblock polymer and the pore size of the dialysis membrane. Larger polymers tend to require shorter dialysis times than smaller diblock polymers.

[0102] Typical solutions of both the diblock and the metal ion solution might be 1 to 100 mM in concentration. A buffer may also be used, such as sodium acetate.

[0103] Nanoparticles can be allowed to form for a prolonged period until a steady state is reached. That could take up to two weeks.

[0104] Alternatively, the nanoparticles might be formed by supplying a homogeneous metal ion source, such as a solution of metal ions, in a process colloquially known as “internal gelation”. The diblock polymers can be dissolved in a saline solution and subsequently contacted with a metal ion complex, e.g. CaEGTA (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid). Nanoparticles are formed due to the homogeneous release of calcium ions from e.g. CaEGTA by a slow change in pH induced, for example, by the introduction of GDL (gluconodelta lactone).

[0105] Diblocks of the invention form well-defined core-shell micelle-like nanoparticles by the introduction of calcium ions, e.g. by dialysis. The core shell particles have a strict phase separation between the G-based core and the dextran corona.

[0106] Metal ions which can be coordinated are preferably multivalent, preferably trivalent or especially divalent. The use of group II metal ions, especially Ca, Ra, Sr and Ba ions is preferred. Other metals of interest include actinides and lanthanides such as yttrium, terbium, lutetium and actinium or some transition metals such as Cu and Zr. Cu-64 and Cu-67 are interesting alternatives for example along with terbium 149 / 152 / 155 / 161. In particular, radionuclides can be coordinated in the nanoparticles of the invention. Suitable radionuclides include those of actinium, thorium, radium, lutetium, gallium, technetium, bismuth, palladium, lead, samarium, iridium, astatine, rhenium, erbium, zirconium and indium.

[0107] Specific radionuclides include actinium-225, thorium-227, radium-223 / 224, lutetium-177 gallium-68, technetium-99, Bismuth-213, gallium-67 / 68, Samarium- 153, Astatine-211 , yttrium-90, Rhenium-186 / 188, erbium-169, zirconium-89, palladium-103, iridium-192 and lead-212, and indium-111 . Terbium 161 and terbium 149 are also important. Radioactive ions that target cancer are of particular interest. The use of Cu-64, Cu-67, Lu-177 and Pb 212.

[0108] The invention may be used to prepare a theranostic pair. For example, Cu- 64 and Cu-67 functionalised NPs might offer such a pair. Other options might be like 68Ga-PSMA for imaging and 177Lu-PSMA for prostate cancer therapy.

[0109] In the case of alginates, the strong and specific interactions of G-blocks with Ca, Ra, Sr and Ba ions could be balanced by steric (repulsive) interactions brought by a neutral polymer block such as dextran conjugated to the G block.

[0110] Nanoparticles preferably have a diameter of 10 to 100 nm, such as 20 to 80 nm. In one embodiment, nanoparticles of the invention are 50 nm or less in diameter. Without wishing to be limited by theory, we perceive that the nanoparticles will comprise a structure like an egg box where the meal ions sit in the cavities of the egg box and the diblock polymers of the invention form the box.

[0111] Stability

[0112] One important consideration is the long term stability of the NPs. It has been found that if barium and strontium are used as metal ions, optionally alone or more preferably in combination with other metal ions, especially other alkaline earth metal ions such as calcium ions, improvements in stability occur. Moreover, Ba and Sr ions appear to be more readily taken up during NP formation. This means therefore that Ba and Sr ions might displace calcium ions already present within the NPs. NPs made with calcium ions could subsequently therefore be exposed to Ba and Sr ions where a displacement reaction would occur. In effect therefore the NPs can be doped with Ba and Sr ions. This same technique could be used for other ions such as radionuclides. Viewed from one aspect therefore, the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises Ra, Ba or Sr ions in combination with other metal ions such as Ca ions.

[0113] It is particularly preferred if NPs contain both Ba / Sr ions and Ca ions, e.g. where the Ca ions are in excess.

[0114] Ba or Sr ions can be present during NP formation or can substitute for ions already present in the NPs. It is also envisaged that NPs comprising Ba or Sr ions offer a narrower NP size distribution than ones made using calcium alone. In particular, NPs saturated with Ba or Sr ions offer very narrow NP size distribution. The size distribution may be such that substantially all NPs have a diameter in the range of 10 to 65 nm.

[0115] Radiolabelling

[0116] The concept of doping can also be extended to the introduction of radionuclides or other valuable toxic metal ions. It has been found that NPs can be labelled with radionuclides after formation or during formation. It will be appreciated that many radionuclides have short-half lives and it is important that the radionuclide is made available rapidly to a patient before too much radioactive decay occurs.

[0117] It has been found that radionuclides can exchange for metal ions already present in the NPs. Thus, NPs of the invention can be prepared using less valuable metal ions such as calcium ions. These NPs are stable and can be stored, such as freeze dried until they need to be used. The NPs can be reconstituted in water and then exposed to radionuclides which partially exchange for metal ions present in the NPs. It will be appreciated that often the concentration of radionuclide ions is rather low and hence exchange will only occur in a limited number of the NPs. The result is a radiolabelled NP that can be immediately used in the relevant medical procedure. This process is particularly important where the radionuclide is radium as this is an alkaline earth metal similar therefore to calcium. Radium can exchange for calcium and be held in the NP.

[0118] Viewed from one aspect the invention provides a process for the preparation of a nanoparticle comprising:

[0119] (I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4 such as a polysaccharide or polyalkylene glycol; and subsequently

[0120] (II) contacting said diblock polymer with metal ions to form nanoparticles comprising said metal ions;

[0121] (III) optionally storing, such as freeze drying, and if required reconstituting said nanoparticles;

[0122] (IV) preparing an aqueous solution of said nanoparticles and contacting the same with a radionuclide or Gd ions to form nanoparticles comprising said radionuclide or Gd.

[0123] The step of exchanging the metal ions present in the NPs with the valuable metal ions can be carried out at elevated temperature (such as 30 to 50°C). Conveniently the aqueous solution used is buffered, e.g. to pH of 5 to 7.

[0124] One benefit of the NPs of the invention is that the daughter ions that are formed in the radioactive decay may be retained or trapped by the NPs. For example, as radium decays first to a very short lived noble gas and then to a bismuth species, before stabilizing as lead, which has high affinity for the guluronate block. The NP prevents the daughter ions escaping and therefore maintains the radioactive centre in the NP. This also has the added benefit that the radioactive decay occurs in the same place and hence the therapeutic benefit of the radionuclide is enhanced. As each decay occurs in essentially the same place, the medical benefit of each decay (e.g. in the emission of an alpha particle which kills a cancer cell) is maximised, and off-target effects caused by escaped daughter radionuclides is reduced

[0125] Chelating agents on the nanoparticles

[0126] It may be however that certain interesting radionuclides or other toxic metal ions are not firmly held by the NPs. To bind such ions, such as gadolinium ions, the NP may be functionalised to carry one or more chelators. Such a chelator can be attached to the corona (shell) of the NP using known chemistry, such as click chemistry. A linking group can be used to bind a chelator to the NP. Such a linking group also means that the chelator extends away from the NP surface. Conveniently, a PEG chain can be used and this chain moves the chelator out from the NP corona. The term chelator is used herein to define an organic structure that coordinates to a metal centre, e.g. a multidentate ligand.

[0127] Suitable chelators are well known multidentate ligands like EDTA or DOTA. One metal ion which may be of particular interest in this regard is Gd.

[0128] GdNCT (neutron capture therapy) is a new promising and non-invasive cancer therapeutic technique. A neutron beam source emitting thermal and epithermal neutrons and GdNCT agents are required. Neutron beam sources are relatively well-developed, compared to suitable GdNCT agents. The NPs of the invention may have utility in this field.

[0129] Of course, it may be that certain metal ions are well held in the core of the NP whilst other metal ions are not so well held. Through the use of a chelator, such less well held metal ions can form part of the nanoparticles of the invention.

[0130] It is preferred therefore if the NPs carry a chelator which can coordinate to valuable metal ions, such as metal ions that are not so easy to coordinate in the core of the NP.

[0131] In one embodiment, NPs can be formed using a metal ion such as calcium. A chelator can them be bound to the NPs and the NPs exposed to a more valuable metal ion. In this way, not only might the valuable metal ion exchange for metal ions already present in the NPs, but a stronger coordination of the valuable metal ion might be achieved through the chelation of the valuable metal ion with a suitable chelator which is carried on the NP, for example on the corona of the NP.

[0132] Specific cell targeting ligands can be attached to the NPs The valuable metal ions such as radionuclides or Gd ions often need to be targeted to specific areas of interest. In a further embodiment, NPs can be functionalised with moieties that target that area of interest so that the NPs accumulate in the area of interest within the body. For example, Gd carrying NPs might be functionalised with cancer targeting molecules that allow higher accumulation of the NPs in cancer cells. Radium carrying NPs might carry a ligand that helps the NPs accumulate in the site of a metastasised prostate cancer in the bone. Any NP of the invention can therefore be functionalised to carry a targeting ligand. The NPs might carry therefore a biological targeting moiety such as prostate specific membrane antigen binder 1 (PSMAi), inhibitors of fibroblast activation protein (FAPi), RGD-peptides (targeting integrins) and folic acid / folate (targeting folic acid receptors), all often overexpressed on cancer cells.

[0133] In a further embodiment therefore a nanoparticle can carry both a chelator and a biological targeting moiety. This can be conveniently achieved, as described below, by combining a mixture of a diblock polymer carrying a chelator and a diblock polymer comprising the biological targeting moiety.

[0134] In one embodiment therefore NPs are functionalised to carry a chelator as herein defined which can coordinate a metal ion, such as DOTA. The NPs also carry a biological targeting moiety such as prostate specific membrane antigen binder 1 (PSMAi), inhibitors of fibroblast activation protein (FAPi), RGD-peptides (targeting integrins) and folic acid / folate (targeting folic acid receptors), all often overexpressed on cancer cells. This results in a flexible NP that in addition to having selective targeting properties, also has the ability to carry alpha / beta- emitting metal ions, such as in DOTA (Pb212, Ac-225, Tb-149, Lu177) all of which have previously been shown to bind DOTA or other chelators.

[0135] If the diblock polymer has bound thereto a chelator then not only can metal ions become bound into the nanoparticle but metal ions can also be chelated by the chelator. This allows therefore post NP formation manipulation of the metal ions in the chelator as certain chelators may have higher affinity for certain metal ions and hence any bound metal ions can be exchanged for higher affinity metal ions. This allows therefore the introduction of challenging metal ions whose concentration is low. This includes therefore radionuclides (Ra, Ac, Cu-64, Pb212) and other toxic ions that might be used in other areas such as contrast agents.

[0136] NPs having bound Gd may offer benefits over current smaller molecule Gd chelates, such as improved contrast, longer imaging times, targeted imaging capabilities, and reduced Gd dose, thereby potentially decreasing concerns related to gadolinium deposition in tissues.

[0137] NPs containing copper 64 may have both therapeutic and diagnostic utility as the metal ion is a beta-emitting and positron-emitting radioisotope. It may have utility in PET-analysis in animals.

[0138] The nanoparticles can therefore be used to administer radionuclides or other interesting metal ions to a patient. They are also a convenient vehicle to store radionuclides. The nanoparticles of the invention are stable under physiological conditions, e.g. at body temperature and pH. They are injectable.

[0139] The diblock polymer can therefore be functionalized to carry a variety of interesting molecules. However it is preferred if a molecule of interest is attached to the corona of the nanoparticles, i.e. the shell of the nanoparticles, i.e. the second component.

[0140] Introduction of chelator and / or biological targeting molecules.

[0141] Conveniently, a molecule of interest can be introduced before or after NP self-assembly, i.e. the NP is first prepared as described herein then a chelator or biological targeting molecule is covalently bound to the NP surface (outer corona) or the chelator or biological targeting molecule is bound to the diblock polymer before NP formation and becomes part of the NP on NP assembly.

[0142] In the first embodiment, the corona forming part of the diblock polymer may already contain a suitable functional group to allow easy binding of a chelator or biological targeting molecule, e.g. an azide for click reactions, after NP formation. This functional group can be present on the diblock polymer before NP formation.

[0143] Alternatively, a molecule of interest is already present on the diblock polymer before NP formation. In one embodiment therefore, a diblock that can form NPs is covalently linked to a chelator or biological targetting molecule on the second component (e.g. dextran), then NPs are allowed to form, e.g. with Ca2+ ions. Thus a chelator (e.g. DOTA) or biological targeting moiety is already attached to the dextran (or any second component before NP formation). For example, NPs carrying cell-specific ligands at the NP surface can be prepared using an oligoguluronate-b-PEG-DOTA) diblock.

[0144] It may be however that forming a nanoparticle with a starting material functionalized to carry a molecule of interest introduces an excessive content of that molecule into the NP. In one embodiment therefore, the invention envisages the formation of nanoparticles from a blend of functionalized and non-functionalised diblock polymers.

[0145] Viewed from another aspect therefore the invention provides a process for the preparation of a nanoparticle comprising:

[0146] (I) obtaining a first diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonate residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4;

[0147] (II) combining said first diblock polymer with a second diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4 and wherein said second diblock polymer additionally comprises a chelator or biological targeting molecule or is functionalised to carry a reactive group to which a chelator or biological targeting molecule can be covalently bound after nanoparticle formation; so as to form a mixture; and subsequently:

[0148] (III) contacting said mixture with metal ions to form nanoparticles; and,

[0149] (IV) if required, reacting said nanoparticles with a chelator or biological targeting molecule so as to covalently bind said chelator or biological targeting molecule to said nanoparticles via said reactive group.

[0150] The invention also relates to nanoparticles obtained by this process. The ratio of non-functionalised and functionalised components can vary however typically the weight ratio of components is such that the chelator / biological targetting molecule functionalised diblock polymer forms 5 to 50 wt% of the blend.

[0151] Of course, it is then possible to prepare nanoparticles from a more complex blend of diblock components where there are different chelators and targeting groups on each diblock polymer. It is simple to prepare a diblock polymer as described herein functionalised to carry a chelator and a separate diblock polymer functionalised to carry a biological targeting molecule. When a blend of these two components is used together, e.g. with a non-functionalised diblock polymer of the invention, the result is nanoparticles functionalised to carry two different ligands.

[0152] It is also possible to prepare a diblock polymer functionalised to carry a chelator and a biological targeting molecule. When a such a diblock polymer is used, e.g. with a non-functionalised diblock polymer of the invention, the result is nanoparticles functionalised to carry two different ligands.

[0153] Viewed from another aspect the invention provides a nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises a chelator such as DOTA and a biological targeting moiety such as prostate specific membrane antigen or folic acid; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

[0154] This technique could be used to generate a wide variety of NPs, e.g. containing both a chelator and biological targeting moiety.

[0155] Another option is to use a blend of an unfunctionalized diblock and one functionalised to carry a reactive group to which a molecule of interest could be bound after NP formation. A diblock polymer functionalised with a biological targetting molecule or chelator could also be present so that the formed NP might contain both a chelator / biological targeting molecule and a functional group capable of post NP formation reaction. The skilled person can therefore devise a variety of different diblock blends to generate many NPs of interest.

[0156] In a further embodiment, the NPs can be prepared from a blend in which a G-block oligomer is present.

[0157] Attaching a chelator such as DOTA to the NPs allows for specific uptake of cations having high affinity for DOTA. Gadolinium (Gd) has a very high affinity for the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). This strong binding is due to the ability of DOTA to form a stable, cage-like structure that encapsulates the gadolinium ion. This high affinity is crucial for the use of Gd-DOTA as a contrast agent in magnetic resonance imaging (MRI) .

[0158] Additional G-blocks

[0159] In one embodiment, in order to enhance the preparation of nanoparticles, an additional oligoguluronate or oligogalacturonate block can be combined with the diblock polymer described herein before nanoparticle formation. Thus 5.0 to 50 wt%, preferably 10 to 40 wt% of oligoguluronate or oligogalacturonate block (based on the weight of blend of the block and diblock polymer) might be combined along with the diblock polymer before nanoparticle formation. Conveniently therefore an aqueous solution of these reactants can be prepared before nanoparticles are formed via addition of metal ions following protocols mentioned above. We have found that if the level of pure G block is to high (more than 50 wt%) then the NPs that form can be too large or gelling occurs.

[0160] In this embodiment, nanoparticles preferably have a diameter of 10 to 200 nm, such as 20 to 150 nm.

[0161] It is envisaged that the inclusion of the oligoguluronate or oligogalacturonate oligomers in combination with the required diblock reduces internal repulsion within the nanoparticle by having dextran-free chains. Reducing steric destabilization of dextran in the nanoparticle core may provide more stable NP's.

[0162] The oligoguluronate or oligogalacturonate blocks used in this embodiment may be those that make up the first component in the diblock polymer and are therefore described in detail above. The oligoguluronate or oligogalacturonate block in this embodiment may have a degree of polymerisation of 5 to 100, such as 10 to 50. Ideally, a G-block is used such as one containing at least 50 mol% of guluronic monomers as described above in the context of the diblock polymer. In one embodiment, the oligomer used can be the same as the first component of the diblock. For example, if the diblock polymer uses a G25 block then the same G25 oligomer could be combined with the diblock polymer to prepare the blend for the preparation of the nanoparticles.

[0163] This oligoguluronate or oligogalacturonate block should not be bound via a linker to a second component. This is unnecessary and would increase cost. The oligoguluronate or oligogalacturonate block should consist of the oligoguluronate or oligogalacturonate block. Alternatively viewed, the oligoguluronate or oligogalacturonate blocks used in this embodiment are monoblocks.

[0164] Ideally therefore if there is a biological targeting moiety or chelator ligand present that should form part of the deblock component. It is possible however for the oligoguluronate or oligogalacturonate monoblocks used in this embodiment to carry a biological targeting moiety or chelator ligand.

[0165] Thus viewed from another aspect the invention provides a process for the preparation of a nanoparticle comprising:

[0166] (I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4;

[0167] (II) combining said diblock polymer with an L-guluronic acid oligomer or galacturonate oligomer comprising at least 50 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation n where n is at least 3 so as to form a mixture; and subsequently:

[0168] (III) contacting said mixture with metal ions to form nanoparticles. The invention also relates to a nanoparticles that are formed in this process. It is most preferred if the process involves: (I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues having a degree of polymerisation n where n is at least 10, such as at least 20; said second component is dextran or polyethylene glycol and has a degree of polymerisation of at least 5, such as at least 10;

[0169] (II) combining said diblock polymer with an L-guluronic acid oligomer comprising at least 50 mol% L-guluronic acid residues having a degree of polymerisation n where n is at least 10, such as at least 20 so as to form a mixture; and subsequently:

[0170] (III) contacting said mixture with metal ions to form nanoparticles, such as calcium ions.

[0171] The presence of these additional G-blocks improves the stability of the nanoparticles subsequently formed. As noted above, the additional G-blocks reduce internal repulsion within the nanoparticle by having dextran free-chains, which reduces steric destabilization of dextran in the nanoparticle core.

[0172] Introduction of metal ions can be achieved via dialysis.

[0173] In one embodiment, different pure G blocks can be added such a G blocks having different degrees of polymerisation. This may enhance the stability of the forming NPs.

[0174] Nanoparticles of the invention, especially those functionalised with relevant biologically active groups and / or chelators / ligands may have medical uses. The nature of that medical use will be dictated by the nature of the groups bound to the nanaoparticle and the nature of the metal coordinated to the nanoparticles. The invention relates however to nanoparticles as hereinbefore defined for use in a method of medical treatment.

[0175] The invention will now be described with reference to the following nonlimiting figures and examples.

[0176] Brief Description of the Figures Figure 1 is a schematic representation of the biosynthesis of functional alginate, partial depolymerization and isolation of pure guluronate blocks (Gn) then terminal conjugation to an activated polysaccharide. .

[0177] Figure 2 shows the reaction of guluronate with PDHA or ADH and subsequent reduction using PB.

[0178] Figure 3 shows DLS analysis of NPs prepared from mixtures of G16-b- PEG5-[click]-PEG5-DOTA (10% and 20% w / w) and G16-b-PEG5-[click]-PEG5-C6- DOTA compared to NPs prepared from pure G16-b-Dex43. Peak sizes: 64 nm (0%), 44 (10%), 41 (20%)..

[0179] Figure 4 is DLS analysis of NPs prepared from mixtures of Gi6-b-PEGs- [click]-PEG22-FA (10%) compared to NPs prepared from pure Gi6-b-Dex43. Peak sizes: 27 nm (0%), 39 (10%), 41 (20%).

[0180] Figure 5. shows residual radioactivity for Ra-223 labelled NPs upon dialysis against 1.2 mM CaCh in 150 mM NaCI. Sample codes:

[0181] 1. Blank

[0182] 2. Diblock control (Gi6-b-Dex4s) (8% M)

[0183] 3. NP Gi6-b-Dex45 prepared by titration 120% (1 / 19 Ba / Ca)

[0184] 4. NP Gi6-b-Dex45 prepared by dialysis 1 mM Ba, 19 mM Ca, 10 mM NaCI

[0185] 5. As 4, containing 10% Gie-b-PEG-FA

[0186] 6. NP Gi6-b-Dex45 prepared by dialysis 0.1 mM Ba, 19.9 mM Ca, 10 mM NaCI

[0187] 7. As 6, containing 10% Gie-b-PEG-FA

[0188] 8. NP G37-b-Dex26 prepared by dialysis 0.1 mM Ba, 19.9 mM Ca, 10 mM NaCI

[0189] 9. As 8, containing 10% G16-b-PEG-FA

[0190] 10. NP Gi9-b-Dex36 (2% M), prepared by dialysis 0.1 mM Ba, 19.9 mM Ca, 10 mM NaCI

[0191] 11. As 10, containing 10% G16-b-PEG-FA

[0192] Figure 6 shows the correlation coefficient and intensity vs time for Gi9-b- Dexse NPs with G19 blocks.

[0193] Figure 7 shows the correlation coefficient and intensity vs time for G39-b- Dex45 NPs with G39 blocks.

[0194] Fig. 8 / 9 shows monitoring of the azide-alkyne Cu-free click reaction between Gie-b-PEGs-Ns and DBCO-PEGs-DOTA by UV spectrometry. The click reaction is demonstrated by the loss of absorbance peaks corresponding to unreacted DBCO. The conjugate is verified by 1 H-NMR (Fig 9). Appearance of a new peak at 5.6-5.8 ppm (marked with circle) is diagnostic for formation of the triazole. Fig. 10a / 10b monitors the azide-alkyne Cu-free click reaction between G37- b-PEGs-Ns and DBCO-PEG k-DOTA by UV spectrometry. The click reaction is demonstrated by the loss of absorbance peaks corresponding to unreacted DBCO. The conjugate is verified by 1 H-NMR (Fig 11). Appearance of a new peak at 5.6-5.8 ppm (marked with circle) is diagnostic for formation of the triazole.

[0195] Figure 12 is the NMR spectrum of purified G16-PEG5-N3.

[0196] Figure 13 is the NMR spectrum of purified Gsy-PEGs-fl-PEGs-DOTA ([] refers to the triazole formed in the azide-DBCO Cu-free click reaction).

[0197] Figure 14 is DLS results obtained after NP formation by dialysis against 20 mM CaCh (in 10 mM NaCI).

[0198] Figure 15 shows LIPLC analysis of Cu-64 labelled nanoparticles. UV detection at 210 and 262 nm and radioactivity detection.

[0199] Figure 16 shows radiochemical yield of Cu-64 labelled NPs obtained by iTLC in citric acid.

[0200] Figure 17 shows radio- and UV-UPLC of DOTA-containing and Cu-64 labelled NPs after incubation in human serum.

[0201] Examples

[0202] Test Methods:

[0203] SEC-MALS

[0204] The molecular weight and intrinsic viscosity of the block polymers (Gn-b-Gnand Gn-b-Dexm) was analysed by Size Exclusion Chromatograph (SEC) with Multiangle Light Scattering (MALS). Samples were dissolved in the mobile phase (0.15 M NaNOs with 10 mM EDTA) and filtered (0.45 pm) prior to injection. Standards were prepared using the same procedure. An Agilent Technologies 1260 IsoPump with a 1260 HiP degasser was used to maintain a flow of 0.5 ml / min during analyses. Samples (0.7 - 1 ml) were injected (50 - 100 pL per injection volume) by an Agiel Technologies Vialsampler. TKS Gel columns 4000 and 2500 were connected in series. DAWN Heleos-ll and ViscoStar II detectors from Wyatt Technology were connected in series with a Shodex refractive index detector (Rl- 5011). Astra 7.3.0 software was used for data collection and processing.

[0205] Preparation of Guluronic Acid Oligomers Guluronic acid oligomers (G oligomer) with different molecular weights and degrees of polymerisation were prepared from extensively hydrolyzed, high guluronate alginate, by acid precipitation to give oligomers with various DPn. DPnwas determined by NMR.

[0206] The guluronic acid oligomers are then activated to form conjugates or combined with activated dextran components to form a diblock polymer.

[0207] Adipic acid dihydrazide (ADH), O,O''-1 ,3,-propanediylbishydroxylamine dihydrochloride (PDHA) and 2-methylpyridine borane complex (a-picoline borane- PB) was purchased from Sigma-Aldrich.

[0208] Preparation of guluronate conjugates - general protocol

[0209] For preparative purposes, oligomers were dissolved in NaAc-buffer (500 mM, pH 4) to a final oligomer concentration of 10 - 20 mM and 10 equivalents PDHA / ADH was added to the reaction. After 24 h, PB (3 - 20 equiv.) was added to the reaction at room temp. The reaction was left for 24 - 120 h with stirring. The reaction mixture was subsequently dialyzed (if DPn < 7 with 100 - 500 Da MWCO and if DPn > 7 with 3.5 kDa MWCO) first against 50 mM NaCI, then against MQ water. Excess linker was removed by semi-preparative SEC, after which samples were dialyzed and freeze-dried. Figure 2 depicts reactions which occur. These conjugates can be combined with the second polymer.

[0210] Comparative Preparation of guluronate diblocks

[0211] Guluronate was dissolved in 500 mM Na-Ac buffer (500 mM, pH 4) to a final concentration of 20 mM. 0.5 equivalents and 6 - 20 equivalents PB was added. Reaction times of 24 h was used for ADH and 120 h for PDHA. The reaction mixture was purified by GFC, dialysis and freeze drying. The guluronate diblock, when exposed to calcium ions, formed a precipitate.

[0212] Preparation of guluronate-Linker-dextran block copolymers - General protocol

[0213] Dextran was activated with 10 equiv. PDHA and purified. Guluronate (2 - 3 equiv.) and Dextran-PDHA was dissolved in NaAc-buffer, after 24 h PB was added (3 - 10 equivalents), and the reaction was left on magnetic stirring for 120 h. The reaction mixture was subsequently dialyzed and freeze dried before purification by semipreparative GFC, dialysis and freeze drying. Particle formation of oligoguluronate-Linker-dextran

[0214] Gn-Linker-Dexm (n = 12 and m = 100) (5 - 10 mg / ml) was dissolved in 1 ml 10 mM

[0215] NaCI and filtered (0.22 pm). After 24 h, the sample was dialyzed (Float-A-Lyzer 100

[0216] - 500 Da) against 20 mM CaCh with 10 mM NaCI (1 - 1 .5 L).

[0217] The Mn, Mw, and DPnfrom SEC MALS analyses of Dexm-b-Gnblock copolymer (after purification by SEC) and the starting material (Gnand Dexm-Linker) is presented in table 1.

[0218] Block copolymer self-assembly in solution

[0219] G4o-linker-Dex o diblock polymer in solution was combined with CaCh (20 mM) introduced into the polymer solution by dialysis. A membrane with a cut-off of 100 - 500 Da was used to minimize the formation of out-of-equilibrium aggregates. After days 10 a steady state had been reached. A population of nanoparticles with diameter around 25 nm corresponds to micellar structures consisting of an alginate- based core hydrogel stabilized by dextran blocks. The hypothesis of a core-shell morphology is supported by the fact that that G40 blocks alone precipitate under similar conditions. Therefore, the diblock structure enabled a strict phase separation between the G-based core and the dextran corona.

[0220] Preparation of nanoparticles (NPs) by dialysis or internal gelation:

[0221] In a further embodiment, nanoparticles can be prepared by dialysis or internal gelation (with CaEGTA or CaCOs / GDL). The two methods give slightly different particles size and also have different kinetics of assembly.

[0222] For these examples a G24-linker-Dex36 diblock polymer was prepared using similar principles to those described above.

[0223] Preparation of NPs by internal gelation: 10 mg G24- DHA-Dex36was dissolved in 1 ml 15 mM NaCI at 22°C and placed on shaking for 12 h. 0.3 ml 100 mM CaEGTA was added and the solution was filtered (0.22 pm). 0.0166 g GDL was dissolved in MQ water, filtered and added immediately to the solution with the diblock. The solution was left at 22°C for 12 h. The formation of nanoparticles was monitored at regular time intervals ( every 1 - 2 h) by dynamic light scattering (DLS) (scattering intensity (kilo counts per second, kcps) and intensity distribution) using ZetaSizer Nano ZS (Malvern Instruments, UK) (25 °C, A = 632.8) with back scattering detection (173°).

[0224] Preparation of NPs by dialysis:

[0225] 10 mg G24- DHA-Dex36was dissolved in 1 ml 10 mM NaCI at 22°C and placed on shaking for 12 h. The solution was filtered (0.22 pm) and transferred to a dialysis bag. Dialysis against 1 L 20 mM CaCh with 10 mM NaCI was continued for 20 h for MWCO > 3.5 kDa, 14 days for 0.5 kDa < MWCO < 1.0 kDa and 14 days for MWCO < 0.5 kDa. The formation of nanoparticles was monitored by dynamic light scattering (DLS) (scattering intensity (kilo counts per second, kcps) and intensity distribution) using ZetaSizer Nano ZS (Malvern Instruments, UK) (25 °C, A = 632.8) with back scattering detection (173°).

[0226] Scheme 1 shows the reactions which occur:

[0227] Nanoparticle formation by Ca, Ba titration A viable alternative to NP formation by dialysis is the direct titration with Ca, Sr, Ba, or mixtures thereof.

[0228] Gi6-b-Dex32 or Gi6-b-Dex43 diblocks were dissolved in pure water (8 mg / ml) and added to a solution containing 3.75 mM CaCh and 3.75 mM BaCh with or without 10 mM NaCI. Solutions were analysed by DLS after 1 h and 24 h. DLS data after 24 hrs shows in all cases NP formation with peak diameters in the range 20-40 nm (Table 2). The last two samples are titrated with Ba alone.

[0229] Table 2

[0230] Saturation refers to the amounts of cations added to balance the negative charge on the alginate. 100% is a stoichiometric amount. For 75% saturation, a sub- stochiometric amount of cations is added.

[0231] Uptake of Ba in NPs

[0232] The uptake of Ba in NPs has not been determined in the literature. To quantify the uptake in NPs an experiment was carried out with Gi6-b-Dex45 diblock. The diblock was dialysed against 20 mM CaCh containing 0.0002, 0.002 or 0.02 mM BaCh until NPs were formed. The NPs were further dialysed against pure water and finally 0.2 M HNO3 to release bound Ba and Ca, which was quantified by ICP-MS. The amount of bound Ba expressed as the mole fraction (XBa) as a function of the Ba / Ca (= R) ratio during the dialysis was determined. Data shows that bound Ba is concentrated 40-70 times in the NP compared to the outer solution.

[0233] NP containing mixtures of diblocks (Gm-b-Dexn) and G-blocks (Gm).

[0234] NPs were prepared from solutions of diblocks (Gm-b-Dexn) containing 0 - 50% (w / w) of pure G-blocks. The total concentration was 4 mg / ml. In more detail, Diblocks Gi9-b-Dex36 and G39-b-Dex45 and the corresponding pure G-blocks of the same lengths as in the diblocks (G19 and G39) were dissolved in MQ (8 mg / ml) and filtered (0.22 pm). Diblocks and G-blocks were combined with varying amounts of G-blocks (0, 10, 25 and 50 wt%) to give a total concentration of 4 mg / mL.

[0235] NPs for the different combinations were prepared by dialysis:

[0236] Gi9-based NPs: Dialysis against 20 mM CaCh in 10 mM NaCI

[0237] G39-based NPs: Dialysis against 5 mM CaCh in 10 mM NaCI

[0238] Dialysis proceeded overnight and was followed by DLS analysis. Properties of some of the NPs formed are presented in Table 3.

[0239] Table 3

[0240] For Gi9-b-Dex36 adding 25% pure G19 resulted in well-defined NPs with elevated radius (35 to 54 nm) but no signs of larger aggregates. Increasing to 50% G19 resulted in gel formation and could not be analysed by DLS. For G39-b-Dex45 adding 10% pure G39 also resulted in well-defined NPs with radius 101 nm but with no signs of larger aggregates. Increasing to 50% G19 resulted in slight (visible) precipitation and a bimodal distribution detected by DLS. The larger species corresponded to particles of several hundred nanometers. They accounted for the majority of the scattering intensity but not necessarily for the volume (or mass) fraction since scattering is proportional to the sixth power of the particle radius. Results are depicted in figures 6 and 7.

[0241] Functionalisation of diblock polymers with a terminal chelator (DOTA)

[0242] G-blocks were conjugated to aminoxy-PEGs-azide by reductive amination to form Gm-b-PEG3-N3blocks. The blocks were purified by gel filtration and dialysis against pure water, and finally freeze-dried.

[0243] Gm-b-PEGs-Ns (m = 13, 16 and 37) blocks were mixed with DBCO-PEGs- DOTA at 40°C to allow for specific azide-alkyne click coupling to form Gm-b-PEGs- [click]-PEGs-DOTA. The reaction was monitored by UV spectrometry (Fig. 8). The G37-b-PEG5-[click]-PEGs-DOTA conjugate was further characterised by NMR (Fig 9).

[0244] Functionalisation of diblock polymers with a terminal ligand (FA)

[0245] Gie-b-PEGs-Ns and Gsyb-PEGs-Ns diblocks (prepared as described above) were reacted with DBCO-PEG22-FA (dissolved in DMSO) to allow for specific azidealkyne click coupling. Excess (unreacted) DBCO-PEG22-FA was extracted with DMSO.

[0246] Gsyb-PEGs-Ns diblocks were reacted with 2 equivalents of DBCO-PEG10k-FA (PEG with 10 kDa molecular weight, corresponding to DP of 227). Excess (unreacted) DBCO-PEG k-FA was extracted with DMSO. The reaction was monitored using UV spectrometry (Figure 10a / b). The conjugates were further characterised by 1 H-NMR (Fig. 11).

[0247] NPs based on multiple different diblock types (heteroparticles) a) NPs with chelator DOTA

[0248] Gi6-b-Dex45 diblocks were mixed with Gi6-b-PEG5-[click]-PEGs-DOTA (0, 10, 20% w / w) (total concentration 4 mg / ml) and dialysed overnight (3.5 kDa cut-off) against 20 mM CaCh in 10 mM NaCI. NP formation was confirmed by DLS. (Fig. 3). No signs of remaining non-associated diblock were observed. b) NPs with ligand (FA)

[0249] Gi6-b-Dex45 diblocks were mixed with Gi6-b-PEGs-[click]-PEG22-FA (0, 10, 20% w / w) (total concentration 4 mg / ml) and dialysed overnight (3.5 kDa cut-off) against 1 mM BaCh,19 mM CaCh in 10 mM NaCI. NP formation was confirmed by DLS. (Fig. 4). No signs of remaining non-associated diblock were observed. c) NPs with both chelator (DOTA) and ligand (FA)

[0250] Mixtures of Gi6-b-Dex45 diblock, Gi6-b-PEG5-[click]-PEGs-FA (10% or 20% w / w) and Gi6-b-PEG5-[click]-PEGs-DOTA (10% or 20% w / w) (total 4 mg / ml) (all dissolved in 10 mM NaCI) were dialysed overnight (3.5 kDa cut-off) against 20 mM CaCh in 10 mM NaCI. No signs of remaining non-associated diblock were observed. Peak size (for 20 wt% FA and 10 wt% DOTA) was 34 nm.

[0251] Adding chelators to preformed NPs by click chemistry (post-labelling):

[0252] Mixtures of Gi6-b-Dex43 diblock and Gie-b-PEG-Na (20% w / w) (total 4 mg / ml) were dialysed overnight (3.5 kDa cut-off) against 20 mM CaCh in 10 mM NaCI. To 100 ul (400 ug) NPs was subsequently added 20 ul of DBCO-PEGs-DOTA labelled with Cu-64 (1.2 MBq / ml). Total radioactivity per sample 24 kBq. After 48 hours, considerable radioactivity remained suggesting high coupling.

[0253] Adding chelators to preformed NPs by click chemistry (post-labelling): UV monitoring NPs containing Gie-b-PEGs-Na and Gi6-b-PEG22-FA (total 1 mg / ml, molar ratio 1 :1.3) were prepared by dialysis overnight (3.5 kDa cut-off) against 20 mM CaCh in 10 mM NaCI.

[0254] The NPs were further reacted with DBCO-PEGs-DOTA (dissolved in DMSO). The click reaction was monitored by UV spectroscopy for 3 days. The decrease in UV absorbance demonstrates that reaction occurs and that DOTA is thus attached to the NP.

[0255] NP Stability - G-block length

[0256] Gm-b-Dexn diblocks with different chain lengths were prepared. Solutions (4 mg / ml) were dialysed overnight (3.5 kDa cut-off) against 20 mM CaCh in 10 mM NaCI. The Ca2+-induced self-assembly was monitored by dynamic light scattering (DLS).

[0257] Samples were subsequently dialysed further against 150 mM NaCI without or with 1.2 mM CaCh (physiological saline) and monitored by DLS. Data are summarised in Table 4.

[0258] Table 4

[0259] Gi2-b-Dex52: NPs remained stable up to one month in 20 mM CaCh in 10 mM NaCI.

[0260] Gi9-b-Dex4s: NPs were stable for over 6 days when dialysed against 150 mM NaCI containing 1.2 mM CaCh.

[0261] Gi9-b-Dex43: These NPs were tested only dialysed against 150 mM NaCI without Ca the NPs remained stable when tested after 24 h.

[0262] NP stabilisation by Sr and Ba

[0263] Gi2-b-Dex52: NPs were prepared by dialysis against either: 20 mM CaCh,

[0264] 19 mM CaCh containing 1 mM BaCh,

[0265] 20 mM BaCh, or

[0266] 20 mM SrCh, all in 10 mM NaCI. All systems formed well-defined NPs. The NPs were further dialysed against 150 mM NaCI containing 1.2 mM CaCh. When monitored after 24 h only minor changes in NP dimensions were observed, but after 48hr NPs without Sr or Ba had disintegrated.

[0267] Gi6-b-Dex43: NPs were prepared by dialysis against either

[0268] 20 mM CaCh,

[0269] 15 mM CaCh containing 5 mM BaCh,

[0270] 10 mM CaCh containing 10 mM BaCh or

[0271] 20 mM BaCh, all in 10 mM NaCI. All systems formed well-defined NPs. The NPs were further dialysed against 150 mM NaCI without CaCh. When monitored after 24 h only minor changes in NP dimensions were observed. None had disintegrated despite the absence of Ca.

[0272] Gi6-b-Dex4s: NPs were prepared by dialysis against either

[0273] 19.9 mM CaCh containing 0.1 mM BaCh or

[0274] 19 mM CaCh containing 1 mM BaCh, both in 10 mM NaCI. Both systems formed well-defined NPs. The NPs were further dialysed against 150 mM NaCI containing 1.2 mM CaCh. When monitored after 24 h none had disintegrated.

[0275] G37-b-Dex26: NPs were prepared by dialysis against either

[0276] 19.9 mM CaCh containing 0.1 mM BaCh or

[0277] 19 mM CaCh containing 1 mM BaCh, both in 10 mM NaCI.

[0278] Both systems formed NPs with larger sizes and broader distributions that the other systems. The NPs were further dialysed against 150 mM NaCI containing 1.2 mM CaCh. When monitored after 24 h none had disintegrated. This data demonstrates physiological stability of the nanopartilces for at least 24 hrs. The term “Not disintegrated” implies that the DLS data (a measure of diameter or size distribution) show no signs of free diblocks (DLS intensity peak under 10 nm).

[0279] Stability - NP with both DOTA and FA

[0280] A mixed NP type containing Gi6-b-Dex43 (70% w / w), Gi6-b-PEGs-[click]-PEG22-FA (20% w / w) and Gi6-b-PEG5-[click]-PEGs-DOTA (10% w / w) was prepared by dialysis against 20 mM CaCh in 10 mM NaCI. It was further dialyzed against 1.2 mM CaCh in 150 mM NaCI. The system showed by DLS no signs of free diblocks after NP formation and remained essentially unaltered for up to 18 days in physiological saline.

[0281] Dialysis, freeze-drying and reconstitution

[0282] NPs were prepared from Gi9-b-Dex36 dialysis against 0.1 mM BaCh and 19.9 mM CaCh in 10 mM NaCI. It was stored for two weeks and then purified by SEC. The NP fraction was further dialyzed against pure water, freeze-dried, and reconstituted by adding pure water to a final concentration of 4 mg / ml. DLS showed that NP dimensions were practically the same after reconstitution. None had disintegrated.

[0283] The experiment included another NP prepared by including 10% (w / w) Gie-b-PEGs- [click]-PEG22-FA during NP formation. The NP fraction was further dialyzed against pure water, freeze-dried, and reconstituted by adding pure water to a final concentration of 4 mg / ml. DLS showed a normal NP without signs of dissolution.

[0284] NPs containing Ra-223: Retention study

[0285] NPs were obtained for a range of diblock combinations by dialysis against 20 mM CaCh in 10 mM NaCI. The NPs were subsequently dialysed against pure water and freeze-dried. NPs were reconstituted by adding pure water to a final concentration of 4 mg / ml and then heating for 60 min at 40’C. Samples (10 ul) were incubated with 750 kBq of Ra-223 dissolved in 10 mM ammonium acetate pH 5.5 and further transferred to separate dialysis membrane devices (Slide-a-Lyzer, 10 kDa membrane). Samples were dialysed against a large volume of 1.2 mM CaCh in 150 mM NaCI. Samples (2.0 ul) were taken at various intervals and assayed for residual (non-dialysable) radioactivity. Results are given in Fig. 5.

[0286] Data show that the ability to first bind and then retain Ra under physiological conditions depends strongly on the type and composition of the NPs. Samples 1 (blank) and 2 (diblock control) both show fast and immediate removal of Ra-223. Samples 4 and 6 also indicate little bound Ra-223, but analogous NPs containing additionally FA (by addition of the diblock Gi6-b-PEG-[click]-PEG-FA) (samples 5 and 7) both showed slow release of Ra-223. NPs prepared from slightly longer G- blocks (Gi9-b-Dex36) with only 2% residual M (Samples 10 and 11) retain about 40% or Ra-223 after 24 h. Best results were obtained for even longer G-blocks (G37-b-Dex26) (samples 8 and 9), with about 60% retention.

[0287] NPs containing active metal, Cu-64 as model for Gd:

[0288] NPs containing DOTA at the NP surface were prepared by co-assembly of a mixture containing the diblocks Gi6-b-Dex43 (100, 90, 80% w / w) and Gi6-PEGs-[]- PEGs-DOTA. Here [] refers to azide-cyclooctyne (DBCO) click. The latter was prepared in two steps: a) Gn(various values of n) were reacted with excess oxyamine-PEGs-Ns by reductive amination. The Gn-PEGs-Ns conjugates were purified with extraction of unreacted oxyamine-PEGs-Ns or by gel filtration and dialysis. The structure was verified by1H-NMR (Fig. 12, n = 16). b) Gn-PEGs-Ns (various values of n) conjugates were further reacted with an excess of DBCO-PEGs-DOTA to form Gn-PEG5-[]-PEGs-DOTA. For structural verification Gsy-PEGs-fl-PEGs-DOTA, was prepared, purified and verified as described above (Figure 13).

[0289] Diblock mixtures were further dialysed for 24 h against 20 mM CaCh in 10 mM NaCI to for nanoparticles. The NPs were analysed by DLS (Fig. 14). The mixtures formed NPs with z-average diameters in the range 35-48 nm.

[0290] The presence of metal-reactive DOTA in the NPs was assayed by reacting the NPs with Cu-64. 200 pL DOTA-NPs (20 w% Gi6-[]-DOTA, 4 mg / mL total) were radiolabelled with 23 pL64CuCh (8.6 MBq / mL) and incubated at 40°C for 20h. The labelled NPs were first analysed by iTLC. No radioactivity corresponding to free Cu- 64 was detected outside the non-migrating starting peak, indicating all Cu-64 was bound.

[0291] The Cu-64 labelled NPs were further assayed by LIPLC using a Superdex 200 increase column eluted with 50 mM CaCh, 0.1 mL / min and monitored by dual wavelength UV and radiodetection. The elution behaviour (retention times) of the individual NP components had been previously determined for the same column using a HPLC set-up containing a refractive index and multiple laser light scattering detector. Results from LIPLC are given in Fig. 15.

[0292] The NP fraction eluting at 10 min shows the highest UV absorption at 262 nm for the DOTA-containing NPs compared to DOTA-negative controls. The NP fraction also retained significant radioactivity assigned to the Cu-64 uptake via DOTA.

[0293] Cu-64 (1.75-28 MBq) was added to 6 x 50 pg NP) and diluted to 57.5 uL with 10 mM AmAc pH 5.8. The samples were incubated at 40°C and analysed by iTLC (citric acid,) after 1h and 20 h. The radiochemical yield (RCY) was determined. Results (Fig. 16) show that up to 15 MBq of Cu-64 can be labelled to 50 ug NPs containing DOTA.

[0294] The results shown above demonstrate that NPs contain DOTA, enable binding of Cu-64. This shows that these NPs can be labelled with metal ions having sufficient affinity for DOTA, including Gd. The literature reports a binding constant of 1022 19for Gd-DOTA at pH 7.

[0295] Serum stability of DOTA-containing NPs

[0296] Human serum (100 pL) was added to 100 pL of DOTA-containing and Cu- 64 labelled NPs (prepared as described above) and incubated at 37°C for 1h. 120 pL of the mixture was diluted with 100 pL PBS and monitored by radio- and UV HPLC (50 mM CuCh, 0.1 mL / min) for up to 24 hours incubation. Results are given in Figure 17. Most of the radioactivity, which decays according to the half-life of 12.7 hours, is found in the NP fraction. Hence, NPs are stable in serum even after Cu-labelling.

Claims

Claims1 . A nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises a chelator such as DOTA; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

2. A nanoparticle as claimed in claim 1 comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4; wherein said nanoparticle comprises a chelator such as DOTA and a biological targeting moiety such as prostate specific membrane antigen or folic acid; wherein said nanoparticle comprises at least one metal ion such as a radionuclide or Gd ions.

3. A nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4;wherein said nanoparticle comprises Ra, Ba or Sr ions in combination with other metal ions such as Ca ions.

4. A nanoparticle as claimed in any preceding claim wherein the second polymer is an oligo or polysaccharide, poly(meth)acrylate or polyalkylene glycol, especially an oligo or polysaccharide.

5. A nanoparticle as claimed in any preceding claim wherein the second polymer is a dextran.

6. A nanoparticle as claimed in any preceding claim wherein the L-guluronic acid oligomer has a degree of polymerisation n of 7 to 70, such as 20 to 40.

7. A nanoparticle as claimed in any preceding claim wherein the second polymer has a degree of polymerisation of 8 to 180, such as 40 to 100.

8. A nanoparticle as claimed in any preceding claim wherein the linker is one that results from an amination, reductive amination or click chemistry.

9. A nanoparticle as claimed in any preceding claim wherein the linker comprises a triazole, two NH-NH-CO- functional groups or two -N-O-CH2- functional groups.

10. A nanoparticle as claimed in any preceding claim wherein the metal ions are calcium ions, optionally in combination with Ac, Y, Lu, Cu, Ca, Sr, Ba or Ra ions or mixtures thereof, in particular copper, Ra, Tb, Gd, lead or Lu, such as Cu-64, Pb212, Ra-223 or Lu 177.

11. A nanoparticle as claimed in any preceding claim being a core shell nanoparticle said first component forming the core and said second component forming the shell of said nanoparticle.

12. A process for the preparation of a nanoparticle comprising:(I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4 such as a polysaccharide or polyalkylene glycol; and subsequently(II) contacting said diblock polymer with metal ions to form nanoparticles comprising said metal ions;(III) optionally storing, such as freeze drying, and if required reconstituting said nanoparticles;(IV) preparing an aqueous solution of said nanoparticles and contacting the same with a radionuclide or Gd ions to form nanoparticles comprising said radionuclide or Gd.

13. A process for the preparation of a nanoparticle as claimed in claim 12 wherein the diblock polymer is functionalised to carry a chelator such as DOTA.

14. A process for the preparation of a nanoparticle as claimed in claim 12 or 13 wherein the radionuclide is an ion of copper, lead or Lu, such as Cu-64, Pb212 or Lu 177.

15. A process for the preparation of nanoparticles comprising:(I) obtaining a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4, such as a polysaccharide or polyalkylene glycol;(II) combining said diblock polymer with an L-guluronic acid oligomer or galacturonic acid oligomer comprising at least 50 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation n where n is at least 3 so as to form a mixture; and subsequently:(III) contacting said mixture with metal ions to form nanoparticles.

16. A process for the preparation of nanoparticles as claimed in claim 15 wherein the content of said L-guluronic acid oligomer or galacturonic acid oligomer is 5 to 40 wt% based on the weight of the diblock polymer and L-guluronic acid oligomer or galacturonic acid oligomer combined.

17. A process for the preparation of nanoparticles as claimed in claim 15 or 16 wherein the L-guluronic acid oligomer or galacturonic acid oligomer is not bound to a second oligomer / polymer such as PEG, preferably is not bound to any second component.

18. A process for the preparation of nanoparticles comprising:(I) obtaining a first diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4;(II) combining said first diblock polymer with a second diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues or at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3;said second component is a second polymer having no more than 30 mol% L-guluronic acid residues or galacturonic acid residues and having a degree of polymerisation of at least 4 and wherein said second diblock polymer additionally comprises a chelator or biological targeting molecule or is functionalised to carry a reactive group to which a chelator or biological targeting molecule can be covalently bound after nanoparticle formation; so as to form a mixture; and subsequently:(III) contacting said mixture with metal ions to form nanoparticles; and,(IV) if required, reacting said nanoparticles with a chelator or biological targeting molecule so as to covalently bind said chelator or biological targeting molecule to said nanoparticles via said reactive group.

19. A process as claimed in claim 18 wherein a third diblock polymer carrying a further different chelator / biological targeting molecule is present in step (II) or the process in step (II) requires a second diblock polymer carrying a chelator / biological targeting molecule and a third diblock polymer functionalised to carry a reactive group capable of reaction after nanoparticle formation.

20. A process as claimed in claim 15 to 19 wherein the nanoparticles are formed via dialysis or exposure of the nanoparticles to a homogeneous source of metal ions, e.g. a solution of metal ions.

21. A process as claimed in claim 19 wherein exposure of the nanoparticles to a homogeneous source of metal ions involves subjecting an aqueous solution of the diblock polymer and positive ions to a change in pH, preferably using GDL.

22. Nanoparticles obtained by a process as described in claim 15 to 21.

23. A nanoparticle comprising a diblock polymer comprising a first component covalently bound via a linker to a second component; wherein said first component is an poly / oligogalacturonate comprising at least 50 mol% galacturonic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer having no more than 30 mol% galacturonic acid residues and having a degree of polymerisation of at least 4;wherein said diblock polymer forms a nanoparticle spontaneously from an aqueous solution comprising metal ions, e.g. in a concentration of at least 0.1 mM of metal ions.

24. A nanoparticle which forms spontaneously from an aqueous solution comprising metal ions (e.g. in a concentration of at least 0.1 mM of metal ions); and a blend of a first and second diblock polymer; said first diblock copolymer comprising: a first component covalently bound via a linker to a second component; wherein said first component is an oligomer comprising at least 50 mol% L- guluronic acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second oligomer / polymer having no more than 30 mol% guluronic acid residues and having a degree of polymerisation of at least 4; and wherein said second diblock polymer comprises a first component covalently bound via a linker to a second component; wherein said first component is an poly / oligogalacturonate comprising at least 50 mol% galacturonate acid residues and having a degree of polymerisation n where n is at least 3; said second component is a second polymer / oligomer having no more than 30 mol% galacturonate acid residues and having a degree of polymerisation of at least 4.

25. Use of nanoparticles as claimed in any of claim 1 to 11 or 22 to 24 to deliver a metal ion to a patient or to remove a metal ion from a medium containing said metal ion.

Images