Diblock Polymer

Abstract

A diblock polymer comprises a first component covalently linked to a second component via a linker, said first component being an oligomer containing at least 50 mol % L-guluronic acid residues and having a degree of polymerization n, where n is at least 3, and said second component being a polymer containing no more than 30 mol % L-guluronic acid residues and having a degree of polymerization m, where 9n>=m>=n / 2.

Description

[Technical field]

[0001] The present invention relates to nanoparticles comprising a diblock polymer comprising an oligo- or polyguluronic acid moiety linked to a second polymer moiety, such as an oligo- or polysaccharide or a polyalkylene glycol. The invention further relates to the diblock polymer itself and to the use of the nanoparticles for delivery of metal ions, such as radionuclides, or organic active agents of interest to a patient. Alternatively, the diblock polymers may be used to coordinate metal ions to allow for their removal from a particular environment. [Background technology]

[0002] Alginic acid is an algal or bacterial polysaccharide that is often used in food, medicine, etc. due to its mild and useful gelling properties. Most alginic acids have a high affinity for multivalent cations, e.g. Ca ions, whose binding leads to hydrogel formation. These phenomena are related to the presence in alginic acid of sequences (blocks) of L-guluronic acid (G), which coexist with blocks of D-mannuronic acid (M) and alternating (..MG..) blocks. Figure 1 shows the structure of L-guluronic acid residues present in alginic acid and the theoretical distribution of these units along the alginic acid chain.

[0003] The content and distribution of G depends on the organism from which the alginate is derived and is the result of the action of a family of mannuronan C5 epimerases.

[0004] Alginate itself can be classified as a block polysaccharide, with the length and distribution of the three block types varying due to the inherent compositional heterogeneity of alginate. The multivalent cation-induced gelling properties of alginate and the relationship to alginate structure, sequence, and chain length have been extensively investigated for decades.

[0005] It is well known how (almost) pure G-blocks can be isolated and separated from the parent alginate (the standard method is a combination of partial hydrolysis with dilute acid and fractional precipitation: the G-blocks selectively precipitate when alginate is hydrolyzed at a specific pH (the M and MG blocks are soluble). In contrast, the properties of isolated M and G-blocks, as well as their incorporation into precisely engineered alginate-based block polysaccharides, have been minimally explored.

[0006] We have now determined that nanoparticles can be prepared from precisely engineered alginate-based block polysaccharides in which the G-blocks are linked in any convenient manner to a second polymer, such as an oligosaccharide or polysaccharide. The G-blocks required are those that contain a high percentage of G residues, because these units coordinate metal ions or active agents to enable the spontaneous formation of nanoparticles in solution. Summary of the Invention [Means for solving the problem]

[0007] Viewed from one aspect, the present invention provides a diblock polymer comprising a first moiety covalently attached to a second moiety via a linker; the first component is an oligomer containing at least 50 mol % L-guluronic acid residues and having a degree of polymerization n, where n is at least 3; the second component is a polymer having 30 mol % or less of L-guluronic acid residues and a degree of polymerization m; Here 9n=>m>=n / 2, for example 9n=>m=>n.

[0008] For the avoidance of doubt, when n / 2 is not an integer, the value of n / 2 is rounded to the nearest integer.

[0009] Viewed from another aspect, the present invention provides a diblock polymer comprising a first moiety covalently attached to a second moiety via a linker; the first component is an oligomer containing at least 50 mol % L-guluronic acid residues and having a degree of polymerization n, where n is at least 3; the second component is an oligosaccharide or polysaccharide having 30 mol % or less of L-guluronic acid residues and a degree of polymerization m; Here, 9n=>m=>n / 2, and when n is less than or equal to 20, m is greater than or equal to 20.

[0010] Viewed from another aspect, the present invention provides a diblock polymer comprising a first moiety covalently attached to a second moiety via a linker; the first component is an oligomer containing at least 50 mol % L-guluronic acid residues; the second component is a second polymer having 30 mol % or less of L-guluronic acid residues; The diblock polymer spontaneously forms nanoparticles in an aqueous solution containing metal ions at a metal ion concentration of at least 0.1 mM.

[0011] Viewed from another aspect, the present invention relates to a method for the preparation of a diblock polymer as defined hereinbefore, and a compound selected from the group consisting of a diblock polymer ... + or a cation, such as a charged organic compound, is provided.

[0012] Viewed from another aspect, the present invention provides a core-shell nanoparticle comprising a diblock polymer as defined hereinbefore, said first component forming the core of said nanoparticle and said second component forming the shell, Within the core of the nanoparticles are ionically bound cations, for example metal ions and / or charged organic compounds.

[0013] Viewed from another aspect, the present invention provides a method for the preparation of nanoparticles, the method comprising the steps of: (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a second polymer having a linking group adapted to react with the guluronic acid oligomer to form a diblock polymer. Contains or or (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a linking group adapted to react with the guluronic acid oligomer and a second polymer; (III) reacting the guluronic acid oligomer having a linking group with a second polymer to form a diblock polymer; or, (I) obtaining guluronic acid oligomers, for example by hydrolysis of alginic acid in the presence of an acid or a base to form guluronic acid oligomers, and activating said oligomers with functional groups; (II) reacting the guluronic acid oligomer with a second polymer adapted to have functional groups reactive with the functional groups of the guluronic acid oligomer to form a diblock polymer. Including, And then, The method includes contacting the diblock polymer with a cation, such as a metal ion, a proton, or a charged organic molecule, to form a nanoparticle.

[0014] The diblock polymer formed in this manner is preferably as defined hereinbefore.

[0015] It is particularly preferred that contact of the diblock polymer with ions is effected by dialysis or by internal gelation, such as by slowly adjusting the pH to release gelling ions from a suitable salt or ionic complex.

[0016] Viewed from another aspect, the present invention provides the use of nanoparticles as defined hereinbefore for delivering metal ions or charged organic compounds to a patient. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 is a schematic representation of the biosynthesis of functional alginate, partial depolymerization, and isolation of pure guluronic acid blocks (Gn), followed by terminal conjugation to activated polysaccharides. Figure 1 also shows the subsequent dimerization with Ca++ and Gn-L-Dexm to form particles. The formation of these dimers then leads to the formation of nanoparticles. [Diagram 2] FIG. 2 shows the reaction of guluronic acid with PDHA or ADH and subsequent reduction with PB. [Diagram 3] Figure 3 shows the NMR and chemical structure of Dex10-PDHA=G3, where n represents the reduced N-oxide. The figure shows the conjugaton before reduction with a Schiff base. 1H-NMR spectrum of the equilibrated reaction mixture with G3 and PDHA-Dex10 was obtained in 500 mM AcOH[d4]pD4. The resonances due to the (E) / (Z)-oximes of the conjugate are annotated. The structure of the conjugated Gn=b-Dexm is included (=showing the unreduced oxime). For comparison, the 1H-NMR spectrum of purified Dex10-PDHA is included. [Figure 4] FIG. 4 is a theoretical illustration of a core-shell nanoparticle of the invention having a radionuclide coordinated via an antibody either in the core or attached to the shell. [Diagram 5] Figure 5 shows data for the G12-PDHA-Dex100 diblock polymer. Residual (unreacted) G12 was selectively removed by SEC (Figure 5a). SEC-MALLS data for the diblock showed a clear shift in the elution profile compared to the free block (Figure 5b). [Figure 6]FIG. 6 shows that nanoparticles produced by G24-b-Dex36 remain stable (have the same particle size) after various treatments. [Figure 7] FIG. 7 shows G24-b-Dex36 nanoparticle size as a function of pH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to diblock polymers and their ability to form nanoparticles that can coordinate cations, e.g., metal ions or protons, or charged organic compounds, e.g., pharmaceutical agents, to enable delivery of the cations, e.g., metal ions or charged organic compounds, to a patient.

[0019] Surprisingly, by attaching a second polymer, e.g. dextran, to the termini of G-alginate (=oligoguluronic acid = G-block), well-defined and highly stable nanoparticles can be formed when cations, e.g. calcium ions, are contacted with the diblock polymer.

[0020] In contrast, alginate by itself (i.e., without the G-block concentration required in the present invention) tends to form hydrogels in the presence of aqueous metal ions, whereas G-blocks alone form precipitates.

[0021] Although the primary goal of the present invention is nanoparticles that coordinate metal ions, proton coordination is also possible.

[0022] In this embodiment, another chain-chain interaction is involved. Low pH leads to protonation of carboxylic acids (-COO- + H+ = -COOH). The pKa of alginic acid is about 3. Sufficiently below this value, alginic acid no longer coordinates metals and precipitates or forms so-called "acid gels". G-blocks usually precipitate (meaning that they can be isolated). Thus, the diblocks of the present invention can form nanoparticles at low pH, e.g. below 3. This formation is reversible and the nanoparticles redissolve when pH>pKa.

[0023] The present invention requires the combination of a first block (or first component) that is an L-guluronic acid oligomer with a second block (or second component) that is a polymer, such as an oligosaccharide or polysaccharide or a polyalkylene glycol. Ideally, the second polymer is water-soluble. The term water-soluble is used herein to define a material that has a water solubility of at least 10 g / L at 20° C.

[0024] The second polymer should be attached to the end of the L-guluronic acid oligomer, i.e., via a functionality at the end of the L-guluronic acid oligomer. In addition, it is preferred that the second polymer is connected to the L-guluronic acid block through a terminal position. Thus, the diblock can be considered to be "linear", i.e., both blocks are connected through the terminal positions of each respective block.

[0025] Guluronic Acid Oligomers The present invention requires the use of guluronic acid oligomers (G-oligomers) as the first component of the diblock polymer. These oligomers are easily obtained from alginic acid. Since natural alginic acid chains do not contain a sufficient concentration of G residues, the natural alginic acid should be hydrolyzed, for example in acid or base, to produce guluronic acid oligomers with a higher content of guluronic acid residues. The guluronic acid oligomers of interest are L-guluronic acid oligomers.

[0026] The alginic acid for preparing the guluronic acid oligomers is preferably one having a high guluronic acid content. Such alginic acids are known. Different natural alginic acids may be used to produce guluronic acid oligomers with different degrees of polymerization.

[0027] The preferred method for breaking down the native alginic acid chains is acid hydrolysis, using strong acids such as sulfuric or nitric acid. This hydrolysis method can be effected by simply exposing the native alginic acid to an acid or base. Conveniently, this method can be effected at room temperature, although elevated temperatures can also be used. Agitation of the reaction mixture ensures that fractionation occurs efficiently.

[0028] The guluronic acid oligomers used in the present invention may have a degree of polymerization ranging from 3 to 100, such as from 5 to 80, in particular from 10 to 50. A more preferred range is from 10 to 40. In practice, it is difficult to obtain very long G blocks from alginic acid, so the use of shorter blocks with a DP of 32 to 50 is preferred.

[0029] The degree of polymerization can be determined via NMR, which represents the number of all monomeric residues in the oligomer. As noted below, not all of these monomeric residues are guluronic acid, but at least 50% of them must be guluronic acid residues.

[0030] The degree of polymerization can be controlled by the length of the hydrolysis step and the nature of the native alginate undergoing hydrolysis. A longer hydrolysis reaction results in a lower degree of polymerization and vice versa. For the avoidance of doubt, DP = degree of polymerization = number of monomers per chain. For example, the polymers GGGGG, GGGGM, or MGMGM have a DP = 5 (i.e., n = 5).

[0031] The degree of polymerization of the guluronic acid oligomer is generally selected depending on the nature of the coordinated cation and the nature of the second copolymer. When the degree of polymerization of the guluronic acid oligomer is low, the second polymer tends to have a higher degree of polymerization (DP) to ensure the formation of nanoparticles. In general, when the coordinated metal ion is large (e.g., Ba), a lower degree of polymerization may be used than when a smaller metal ion such as Ca is used.

[0032] Alternatively, the weight average molecular weight (Mw) of the guluronic acid oligomers may range from 1000 to 40,000. Mw may be determined using GPC, light scattering, or a combination of both.

[0033] It will be appreciated that guluronic acid oligomers may be prepared from alginic acid by methods known in the art, including hydrolysis, enzymatic degradation (e.g., using lyases), or alkaline beta-elimination. Those skilled in the art will be able to devise suitable methods for forming these oligomers. Although the guluronic acid oligomer may contain several other monomeric residues, it is essential that the guluronic acid content of the guluronic acid oligomer is at least 50 mol%, preferably at least 70 mol%, in particular at least 85 mol%. The intention is to prepare guluronic acid oligomers that contain a significantly higher concentration of guluronic acid than natural alginic acid. The alginic acid is fractionated and oligomers with lower guluronic acid are removed. Only the oligomer blocks with high G content are of interest. High G content improves metal ion binding selectivity.

[0034] Alternatively, 50% or more of the monomer residues of the guluronic acid oligomer are L-guluronic acid, and preferably 70% or more, such as 85% or more of the monomer residues are L-guluronic acid. G The value is greater than or equal to 0.5, e.g. greater than or equal to 0.7, in particular greater than or equal to 0.85. Of course, it is also possible to use pure guluronic acid oligomers (e.g., greater than or equal to 99 mol %, F G0.99). Other residues that may be present in the existing guluronic acid oligomers include mannuronic acid.

[0035] The hydrolysis reaction results in cleavage of the polymer chains, allowing the target guluronic acid oligomers to be fractionated from the mixture of oligomers formed.

[0036] It will be appreciated that a mixture of guluronic acid oligomers may be used when preparing the diblock polymer of the present invention. When natural alginic acid is hydrolyzed to isolate high G content oligomers, such a mixture may be used as the first component of the diblock polymer of the present invention, or further purification may be used to isolate a mixture containing a single oligomer or a smaller number of different oligomers. Those skilled in the art may adjust the nature of the first component of guluronic acid oligomers depending on the properties of the nanoparticles required. However, it is required that the mixture contains oligomers with substantially all components having at least 50 mol% guluronic acid residues.

[0037] Determining the number of repeat units in a guluronic acid oligomer and determining the number of guluronic acid residues in a guluronic acid oligomer can be accomplished using known analytical techniques such as, for example, NMR. MALS, SEC-MALS, and viscosimetry can also be used to determine the Mw of the polymer, and that information can be used to determine the number of repeat units or monomers in the polymer.

[0038] The guluronic acid oligomer must then be linked to a second polymer via any convenient chemical property. The hydrolytic nature of alginic acid means that the guluronic acid oligomer contains a carbonyl group, such as an aldehyde functionality. This carbonyl or specifically aldehyde functionality can be utilized when conjugating the guluronic acid oligomer to the second polymer. This carbonyl functionality is preferably located at the end of the guluronic acid oligomer.

[0039] Linker The guluronic acid oligomer is joined to the second polymer via a linker. The nature of the linker is not critical and the skilled chemist can devise many ways to join the guluronic acid oligomer to the second polymer. In theory, this linker can be a simple one atom, such as an -O- atom, that allows the linking of the two components of the diblock polymer. However, preferably a dedicated linking molecule is used.

[0040] Any suitable covalent chemistry may be used with suitable functionalization of the reactants to create suitable nucleophiles and electrophiles. The use of click chemistry is a particularly preferred method for joining larger molecules. For example, aminooxy-azide reacts easily with aminooxy-DBCO in the well-known click chemistry reaction. Functionalization of the reactants with complementary click groups allows simple connection of the reactants. Thus, the linker in this embodiment becomes the atom between the L-guluronic acid oligomer and the second polymer. Thus, a preferred linker may include a triazole group (formed by the click reaction of an alkyne and an azide).

[0041] The linker of the present invention is preferably multifunctional, for example bifunctional or trifunctional. In one embodiment, a single bifunctional linker is used, i.e., the linker must be capable of reacting with both reactants. The linking of the two components can be effected simultaneously, but more conveniently, one component is reacted with the linker first, and then the other component is reacted with the functionalized component.

[0042] Ideally, the linker is a small molecule with a Mw of less than 300 g / mol, for example 50-200 g / mol. However, larger linking groups can also be used, for example polyalkylene oxide chains. Preferably, such polymeric linkers will have less than 20 repeat units.

[0043] Conveniently, the ligation reaction utilizes terminal masked carbonyl / aldehyde groups of the guluronic acid oligomer and, if present, the second polymer. Ideally, the ligation reaction therefore involves a reaction involving reductive amination, amination, or click chemistry with a functional group selected from, for example, azides, alkynes, thiols, alkenes, etc. The use of dioxyamines or dihydrazides is preferred.

[0044] The linker may thus form a Schiff base (oxime or hydrazone) with the first or second component. Conveniently, one of the components is functionalized with a bifunctional reductive amination type reagent, such as, for example, 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 easily adapted by the skilled chemist.

[0045] Advantageously, the linker is a bifunctional linker, which may include, for example, C 1-10 There are terminal functional groups linked by an alkylene chain, such as a linear alkylene chain. Functional groups of interest include -O-NH2 or -CO-NH-NH2. The length of the linker is an additional tool that a skilled chemist can use to alter the properties of the diblock polymer, as longer linkers can alter the viscosity of the diblock polymer.

[0046] When the reaction is complete, the Schiff base may be reduced (e.g., to form a stable amine). Suitable reducing agents include picoline borane or sodium cyanoborohydride. Such species may be more chemically stable than the oximes or hydrazones.

[0047] In Figure 2, the reaction of guluronic acid with PDHA or ADH is depicted to form an oxime or hydrazone, which is then reduced to an N-oxide or hydrazine. It will be appreciated that the hydrazone has an equivalent form, the pyranoside. Equivalent cyclic forms, such as furanosides, may also exist.

[0048] Ideally, the linker should connect the terminal positions of the guluronic acid oligomer and the second polymer.

[0049] A person skilled in the art could easily devise a suitable chemistry for linking the two components. In one embodiment, the linker may contain 5-20 backbone atoms (i.e., the length of the chain connecting the two blocks is 5-20 atoms). For example, the O-CH2-CH2-CH2-CH2-O linker contains 6 backbone atoms.

[0050] In some embodiments, the linker may comprise a short chain polyalkylene glycol, such as, for example, PEG. Such a chain may have up to 10 repeating units, such as up to 5 such units.

[0051] Second Polymer The second component of the diblock polymer is a polymer such as, for example, an oligosaccharide or polysaccharide, a poly(meth)acrylate, or a polyalkylene glycol. It will be appreciated that the second soluble polymer must be different from the guluronic acid oligomer. Thus, the second polymer does not contain more than 30 mol % of guluronic acid residues. Ideally, the second polymer does not contain any guluronic acid residues. The second polymer is preferably not derived from alginic acid.

[0052] Alternatively, the second polymer does not interact with the cation coordination of the G block.

[0053] The second polymer is preferably a water-soluble polymer. Some insoluble polymers may also be used, especially those with a low degree of polymerization, such as insoluble chitin oligomers with a DP of 6-40.

[0054] The second polymer is one that, when linked to the G oligomer, forms nanoparticles in the presence of cations, e.g., metal ions. Second polymers that form precipitates in those conditions are excluded.

[0055] The second polymer preferably has a weight average molecular weight (Mw) higher than that of the guluronic acid oligomer. Ideally, the Mw of the second polymer is at least twice as high as that of the guluronic acid oligomer, e.g., 3-8 times higher. However, if the Mw of the second polymer is too high (e.g., more than 20x the Mw of the guluronic acid oligomer), it is more likely to form precipitates than target nanoparticles.

[0056] Alternatively, the degree of polymerization of the second polymer should be equal to or greater than the degree of polymerization of the guluronic acid monomer. Thus, the ratio of n to m is important, where n is the DP of guluronic acid 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). A particularly preferred ratio is 4n=>m>=n.

[0057] Thus, in general, precipitation occurs when the DP of G is much larger than the DP of the second polymer. When both oligomers are short, e.g., when the DP of both oligomers is less than 15, and the DP of G is equal to the DP of the second polymer, precipitation occurs rather than NP formation. Thus, when the DP of the G oligomer is in the range of n=3-15, the DP of the second polymer is preferably m=30-180.

[0058] For example, (Ca ions) G 10 -Linker-Dex 40 leads to the formation of nanoparticles, whereas G 10 -Linker-Dex 100will precipitate.

[0059] G 40 -Linker-Dex 40 is G 40 -Linker-Dex 100 Nanoparticles are formed in the same manner.

[0060] If the value of m exceeds 180, there is a risk that the diblock polymer becomes water-soluble, so m is preferably 180 or less.

[0061] The exact values ​​of m and n that result in a precipitate or nanoparticles may vary depending on the nature of the cation coordinated within the nanoparticle.

[0062] Without wishing to be bound by theory, it is believed that an appropriate Mw of the DP of the second polymer promotes the spontaneous formation of nanoparticles in an appropriate medium, which is typically an aqueous medium.

[0063] When both polymers have at least 20 repeat units, the Mw of the water-soluble polymer may be less than that of the guluronic acid oligomer.

[0064] Determining the number of repeat units in the second polymer can be accomplished using well-known analytical techniques, such as, for example, NMR. In addition, MALS, SEC-MALS, or viscosity measurements can be used to determine the Mw of the polymer, and that information can be used to determine the number of repeat units (monomers) in the polymer. Many commercial polysaccharides are available on the market with defined degrees of polymerization.

[0065] In effect, it can be considered that the water-soluble polymer forms the shell of the core-shell nanoparticle and the guluronic acid oligomer forms the core. The nanoparticles can therefore be considered as micelles or polymersomes.

[0066] Preferred water-soluble polymers are polyethylene glycol or oligo- or polysaccharides, in particular hyaluronan, pullulan, β-1,3-glucan, heparin, glycosaminoglycans, amylose, chitosan, or dextran. Dextran is a branched poly-α-D-glucoside of microbial origin, with mainly C-1→C-6″ glycosidic bonds. The dextran chains are of various lengths.

[0067] The water-soluble polymer may be functionalized to have a linker as previously described herein, followed by linking reaction of the guluronic acid oligomer with the water-soluble polymer.

[0068] When the second component is a polyalkylene glycol, it ideally contains at least 10 repeat units.

[0069] In a highly preferred embodiment, the guluronic acid oligomers are linked to the dextran, ideally via reductive amination, ie the linker contains an N-oxide or a hydrazine.

[0070] Diblock Polymer Thus, the engineered diblock polymers of the present invention comprise, e.g., consist of, two or more different blocks linked by a suitable conjugation method. The diblock polymers of the present invention may be linear.

[0071] The diblock polymer of the present invention is referred to herein as G n The diblock polymers can be named G-L-xxx, where G is a guluronic acid oligomer with a degree of polymerization n. L is a linker and xxx is a second polymer, such as dextran. In particular, diblock polymers can be named G n -L-Dex m where Dex is dextran and m is the degree of polymerization of dextran.

[0072] 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.

[0073] The ratio of n to m is also important. Ideally, the ratio is 2:1 to 1:9. Therefore, it is preferable that 9n>m>n / 2. The particularly preferable ratio is 4n=>m>=n.

[0074] Nanoparticles The diblock polymers of the present invention self-assemble under defined conditions, where one block can undergo short-range attractive interactions, whereas the other block undergoes long-range repulsive interactions. Self-assembly is a spontaneous process that results in a great variety of structures, the characteristics of which depend on the molecular parameters of the starting block polymer. The diblock polymer is preferably dissolved in water. Upon addition of metal ions, nanoparticles are formed. Without being limited by theory, it is believed that the presence of metal ions initially allows the formation of dimers of the diblock polymer. The formation of these dimers leads to the formation of nanoparticles.

[0075] In contrast, when a diblock polymer based on two oligoguronates is used, the addition of metal ions leads to the formation of a solid precipitate rather than nanoparticles.

[0076] Typically, an excess of metal ions is added to ensure nanoparticle formation. The concentration of metal ions required in the solution varies depending on the nature of the metal ions. It will also be appreciated that a mixture of metal ions may be used. Generally, the concentration of metal (2+) ions required in the solution follows the following order: Mg>>Mn>Ca>Sr>Ba>Cu>Pb. In some embodiments, a saturated solution may be used.

[0077] The addition of metal ions to an aqueous solution of the diblock polymer allows for the spontaneous formation of the nanoparticles of the invention, ideally by using dialysis or internal gelation.

[0078] In the internal gelation method, metal ions, e.g., Ca, are first dispersed within alginate, e.g., as metal carbonate microparticles or as soluble metal complexes, e.g., metal-EGTA or metal-EDTA complexes. The pH is slowly lowered, using a pH modifier, e.g., GDL, sufficiently to release the metal ions from the source to induce metal-alginate gelation. In the presence of the diblocks of the present invention, this method surprisingly results in stable nanoparticles. When alginate is used, a hydrogel is formed.

[0079] Conveniently, dialysis involves dialyzing the diblock solution against a metal ion solution, such as a solution of Ca ions, e.g., CaCl2. The length of 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.

[0080] Typical solutions of both the diblock and metal ion solutions may be at concentrations of 1-100 mM. Buffers such as sodium acetate may also be used.

[0081] Nanoparticles can form over an extended period of time until a steady state is reached, which can be up to two weeks.

[0082] Alternatively, nanoparticles may be formed by providing a homogeneous source of metal ions, e.g., a solution of metal ions, in a method colloquially known as "internal gelation." The diblock polymer may be dissolved in saline and then contacted with a metal ion complex, e.g., CaEGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid). Nanoparticles are formed by homogeneous release of calcium ions, e.g., from CaEGTA, by a slow change in pH, induced, e.g., by the introduction of GDL (gluconodelta lactone).

[0083] Oligoguluronic acid-L-dextran diblocks form well-defined core-shell micelle-like nanoparticles upon introduction of calcium ions, e.g., by dialysis. The core-shell particles have a strict phase separation between the G-base core and the dextran corona.

[0084] In contrast, free oligoguluronic acid chains precipitate under the same conditions. This is perhaps the first report of a stimuli-sensitive diblock polysaccharide without lateral modifications.

[0085] Thus, alginic acid, G-blocks, and Gn-b-Xm diblocks react with calcium salts or dilute acids in different ways. That is, alginic acid generally forms macroscopic hydrogels and G-blocks precipitate in solution, whereas Gn-b-Xm diblocks form stable nanoparticles with a core / shell structure. In this specification, the alternative name Gn-b-Xm is used to define a diblock (deblock) with Gn (G-block), b as a linker, and Xm as a second component.

[0086] The metal ions that can be coordinated are preferably multivalent, preferably trivalent or especially divalent. The use of metal ions of group II, 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. For example, Cu-64 and Cu-67 are interesting options, along with terbium 149 / 152 / 155 / 161. In particular, radionuclides can be coordinated in the nanoparticles of the present invention. Suitable radionuclides include those of actinium, thorium, radium, lutetium, gallium, technetium, bismuth, palladium, lead, samarium, iridium, astatine, rhenium, erbium, zirconium and indium.

[0087] 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, rhenium-186 / 188, erbium-169, zirconium-89, palladium-103, iridium-192, and lead-212, and indium-111. Cancer-targeting radioactive ions are of particular interest.

[0088] In the case of alginate, the strong and specific interactions of the G-block with Ca, Ra, Sr, and Ba ions can be balanced by steric (repulsive) interactions provided by neutral polymer blocks, e.g., dextran, conjugated to the G-block.

[0089] The nanoparticles preferably have a diameter of 10 to 100 nm, such as 20 to 80 nm.

[0090] Thus, nanoparticles can be used to administer radionuclides or other metal ions of interest to patients.Nanoparticles are also convenient media for storing radionuclides.The nanoparticles of the present invention are stable under physiological conditions, such as body temperature and body pH.These nanoparticles are injectable.

[0091] It is difficult to prepare nanoparticles containing certain metal ions. For example, it is difficult to form nanoparticles using magnesium ions because magnesium ions do not spontaneously combine with diblock polymers to form nanoparticles. However, it would be useful if nanoparticles containing magnesium could be formed, because such nanoparticles may have a higher affinity for certain targets.

[0092] It has been found that magnesium ions can be introduced into nanoparticles via replacement of metal ions already present in the nanoparticles. Thus, after forming nanoparticles with, for example, calcium ions according to the protocols described herein, the nanoparticles can be exposed to a magnesium ion solution, for example by dialysis with a magnesium ion solution. Additionally, the strength of the magnesium ion solution can be varied to alter the amount of metal ions replaced. By increasing the concentration of magnesium ions in the solution, more metal ions are displaced from the nanoparticles. Our experiments suggest that there is an optimal concentration, above which the replacement is less effective. One skilled in the art can easily determine the concentration required to maximize replacement. Typically, the concentration is 0.05-20 mM. Counterions such as halides, nitrates, etc. are suitable for the metal ion solution. We have demonstrated that 50-95% of the metal ions in the nanoparticles can be replaced, resulting in 50-95% replacement ions, such as Mg ions.

[0093] It will be appreciated that this principle of substitution may be used for a variety of different metal ion combinations to allow for the introduction of different metal ions into the nanoparticles, such as the introduction of alkali metal ions such as sodium or potassium ions.

[0094] Thus, in one embodiment, the method of the present invention further comprises a step in which nanoparticles comprising a first metal ion are combined with a solution of a second metal ion, e.g., nanoparticles comprising calcium ions are combined with a solution of magnesium ions, thereby displacing at least a portion of the first metal ions and replacing them with a portion of the second metal ions.

[0095] In further embodiments, the nanoparticles of the present invention may coordinate charged organic molecules of biological interest, such as charged pharmaceuticals. The guluronic acid core is typically negatively charged and therefore readily coordinates metal ions. The same ionic interactions would be suitable for the coordination of charged organic molecules, such as positively charged organic molecules. Many pharmaceuticals in salt form are charged and therefore suitable for coordination in the nanoparticles of the present invention. Such molecules may be used in place of or in addition to metal ions.

[0096] In addition, the strength of binding to the charged species can be adjusted depending on the G content of the first component. Higher G content tends to result in stronger binding. Therefore, when the physiological release of the charged species is important, the G content of the first component can be reduced to facilitate release.

[0097] In further embodiments, the diblock polymers and nanoparticles may be further functionalized to carry biological targeting compounds, such as antibodies, ligands, etc. This functionalization may be performed before or after nanoparticle formation. These biological targeting molecules may themselves carry drugs of interest. For example, radionuclides may be coordinated to antibodies that are bound to the diblock polymers of the invention.

[0098] In one embodiment, it is considered that nanoparticles containing biological targeting compounds can be formed by incorporating biological targeting compounds, such as peptides, into diblock polymers that become part of the nanoparticles during formation.Alternatively, relevant biological targeting moieties can be combined with G-block polymers that become part of the nanoparticles during formation.For example, diblock polymers that include G-blocks and peptides as defined herein can be combined with diblock polymers of the present invention, such as those that include Gn-b-dextran, and incorporated into nanoparticles when they are formed.

[0099] Thus, for example, the diblock polymer of the present invention, such as Gn-b-dextran, n Nanoparticles containing peptide ligands can be prepared by adding -b-peptide. The ratio in this method can be used to adjust the concentration of biomolecules in the nanoparticles.

[0100] Although we illustrate this concept below with peptides, any suitable biomolecule can be used to attach to the G-block. For example, a targeting ligand can be combined with a guluronic acid oligomer. An example includes folic acid, which can be activated by a click chemistry linker for attachment to the azide bearing G-block.

[0101] Other biomolecules include antibodies, antibody fragments, nanobodies, affibodies, peptides (such as bombesin, octreotide, or RGD), peptidomimetics, aptamers (nucleic acids), small molecules (such as tyrosine receptor inhibitors), hyaluronic acid, and other ligands that target receptors or cell surface molecules that are overexpressed in cells representing diseased tissue.

[0102] It is contemplated that biological moieties attached to G blocks may be combined with the diblock polymers of the present invention to spontaneously incorporate as part of the nanoparticles formed in the presence of metal ions.

[0103] Viewed from another aspect, the present invention provides a method for the preparation of nanoparticles, the method comprising the steps of: (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a second polymer having a linking group adapted to react with the guluronic acid oligomer to form a diblock polymer. Contains or or (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a linking group adapted to react with the guluronic acid oligomer and a second polymer; (III) reacting the guluronic acid oligomer having a linking group with a second polymer to form a diblock polymer; or, (I) obtaining guluronic acid oligomers, for example by hydrolysis of alginic acid in the presence of an acid or a base to form guluronic acid oligomers, and activating said oligomers with functional groups; (II) reacting the guluronic acid oligomer with a second polymer adapted to have functional groups reactive with the functional groups of the guluronic acid oligomer to form a diblock polymer; Including, And then, contacting the diblock polymer with a first cation, such as, for example, a metal ion, a proton, or a charged organic compound, to form nanoparticles; contacting the nanoparticles with a second cation, e.g., a metal ion different from that used in the previous step, such that the second cation at least partially replaces the first cation in the nanoparticles.

[0104] Viewed from another aspect, the present invention provides a method for the preparation of nanoparticles, the method comprising the steps of: (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a second polymer having a linking group adapted to react with the guluronic acid oligomer to form a diblock polymer. Contains or or (I) obtaining guluronic acid oligomers, for example by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a linking group adapted to react with the guluronic acid oligomer and a second polymer; (III) reacting the guluronic acid oligomer having a linking group with a second polymer to form a diblock polymer. or, (I) obtaining guluronic acid oligomers, for example by hydrolysis of alginic acid in the presence of an acid or a base to form guluronic acid oligomers, and activating said oligomers with functional groups; (II) reacting the guluronic acid oligomer with a second polymer adapted to have functional groups reactive with the functional groups of the guluronic acid oligomer to form a diblock polymer. Including, And then, The method includes contacting the diblock polymer with a cation, such as a metal ion, a proton, or a charged organic compound, in the presence of a diblock polymer comprising a guluronic acid oligomer linked to a peptide to form a nanoparticle. EXAMPLES

[0105] Test method: SEC-MALS Size Exclusion Chromatograph (SEC) with Multiangle Light Scattering (MALS) was used to determine the size of the block polymer (G n -bG n and G n -b-Dex m The molecular weight and intrinsic viscosity of the 1,2-dichlorophenyl ether (1,2-dichlorophenyl ether) were analyzed. Samples were dissolved in the mobile phase (0.15 M NaNO3 and 10 mM EDTA) and filtered (0.45 μm) before injection. Standards were prepared using the same procedure. A flow of 0.5 ml / min was maintained during the analysis using an Agilent Technologies 1260 IsoPump and 1260 HiP degasser. Samples (0.7–1 ml) were injected (50–100 μL per injection volume) by an Agilent Technologies Vialsampler. TKS Gel columns 4000 and 2500 were connected in series. Wyatt Technology DAWN Heleos-II and ViscoStar II detectors were connected in series with a Shodex refractive index detector (RI-5011). Astra 7.3.0 software was used for data collection and processing.

[0106] Preparation of guluronic acid oligomers Various DPs were obtained from extensively hydrolyzed high guluronic acid alginates by acid precipitation. n Guluronic acid oligomers (G oligomers) with different molecular weights and degrees of polymerization were prepared by obtaining oligomers with DP n was determined by NMR.

[0107] The following guluronic acid oligomers were prepared: DP21, F G 0.90 (where DP n is the average degree of polymerization, and F G is the fraction of monomers that are guluronic acid, i.e., mol % of guluronic acid). DP4. Sample F G was >0.9. DP10. Sample F G was >0.9. DP11. Sample F G was >0.9. DP12. Sample F G was >0.9.

[0108] The guluronic acid oligomers were then activated to form conjugates or combined with activated dextran moieties to form diblock polymers.

[0109] Adipic acid dihydrazide (ADH), O,O'-1,3-propanediylbishydroxylamine dihydrochloride (PDHA), and 2-methylpyridine borane complex (α-picoline borane-PB) were purchased from Sigma-Aldrich.

[0110] Preparation of Guluronic Acid Conjugates – General Protocol For preparation purposes, oligomers were dissolved in NaAc buffer (500 mM, pH 4) to a final oligomer concentration of 10-20 mM, and 10 equivalents of PDHA / ADH were added to the reaction. After 24 h, PB (3-20 equivalents) was added to the reaction at room temperature. The reaction was left under stirring for 24-120 h. The reaction mixture was then dialyzed (by 100-500 Da MWCO for DPn<7, or 3.5 kDa MWCO for DPn>7) first against 50 mM NaCl and then against MQ water. After removing excess linker by semi-preparative SEC, the samples were dialyzed and lyophilized. Figure 2 shows the reactions carried out. These conjugates can be combined with a second polymer.

[0111] Comparative preparation of guluronic acid diblocks Guluronic acid was dissolved in 500 mM Na-Ac buffer (500 mM, pH 4) to a final concentration of 20 mM. 0.5 equiv. and 6-20 equiv. of PB were added. Reaction times of 24 h for ADH and 120 h for PDHA were used. The reaction mixtures were purified by GFC, dialysis, and lyophilization. The guluronic acid diblock formed a precipitate when exposed to calcium ions.

[0112] Preparation of Guluronic Acid-Linker-Dextran Block Copolymers According to the Invention - General Protocol Dextran was activated and purified with 10 equiv. guluronic acid (2–3 equiv.) and dextran-PDHA were dissolved in NaAc buffer, and after 24 h PB was added (3–10 equiv.), and the reaction was left for 120 h with magnetic stirring. The reaction mixture was then dialyzed and lyophilized before purification by semi-preparative GFC, dialysis, and lyophilization.

[0113] G-linker-dextran particle formation G n -Linker-Dex m (n=12 and m=100) (5-10 mg / ml) were dissolved in 1 ml of 10 mM NaCl and filtered (0.22 μm). After 24 h, samples were dialyzed (Float-A-Lyzer 100-500 Da) against 20 mM CaCl2 and 10 mM NaCl (1-1.5 L).

[0114] Dex m -bG n Block copolymers (after purification by SEC) and starting materials (G n and Dex m -Linker) by SEC MALS analysis of Mn, Mw, and DP n are presented in Table 1.

[0115] [Table 1]

[0116] To prove the concept, further diblock polymers were prepared following the same protocol as above and analyzed using NMR. Shorter chain dextran and guluronic acid oligomers were used to make NMR assignments easier. Figure 3 shows the diblock structure of G3 and PDHA-Dex in 500 mM AcOHd4 pD4. 10 Dex of the equilibrium reaction mixture (1:1) 10 NMR spectrum of b-PDHA-G3 (600 MHz). The resonances due to the (E) / (Z)-oximes of the conjugate are annotated. Conjugated G3=b-Dex m (= indicates the unreduced oxime). For comparison, purified Dex 10 - Includes the 1H-NMR spectrum of PDHA.

[0117] In conclusion, D.P. 100 Dextran chains and DP 10 As demonstrated by the dextran chains, conjugation of oligoguluronic acid with PDHA-activated dextran chains is efficient for longer and shorter chains.

[0118] Self-assembly of block copolymers in solution G in solution 40 -Linker-Dex 100 The diblock polymer was combined with CaCl2 (20 mM), which was introduced into the polymer solution by dialysis. To minimize the formation of out-of-equilibrium aggregates, a membrane with a cut-off of 100-500 Da was used. A steady state was reached after 10 days. A population of nanoparticles with a diameter of about 25 nm corresponds to a micellar structure consisting of an alginate-based core hydrogel stabilized by dextran blocks. The hypothesis of a core-shell morphology is G 40 This is supported by the fact that the blocks alone precipitate under similar conditions, and thus the diblock structure allowed for strict phase separation of the G-base core and the dextran corona.

[0119] Self-assembly of block copolymers in solution Similarly, G11 -b-Dex 100 was prepared. 11 -b-Dex 100 had a significantly different behavior under similar conditions; that is, this block copolymer tended to form larger nanoparticles in solution with Ca (>1000 nm). From a thermodynamic point of view, this could mean that due to the shorter G-block, the entropy loss associated with the formation of the dextran corona is not compensated by a sufficient enthalpy gain due to the gelation of the G-block. Therefore, the length ratio of the two blocks needs to be carefully considered to have self-assembly properties.

[0120] Further Diblock Polymers The high reactivity of oligouronic acids with PDHA suggests that reactions with PDHA-activated oligosaccharides to give diblock oligosaccharides or polysaccharides would proceed with similar results, which was tested in a kinetic study with β-1,3-glucan-PDHA (DP9).

[0121] In addition, for the preparation of symmetric blocks, n Reactions with -PDHA were also investigated. All conjugates (oximes) were fully reduced by picoline borane (PB) prior to conjugation with G3. These PDHA-activated oligosaccharides represent a wide range of different chemical properties (Table 3). Dextran is a neutral chain with high chain flexibility due to α-1,6 linkages. Amylose (α-1,4 linked glucan) and β-1,3-glucan are both semi-rigid neutral chains with the ability to form higher order structures. Collectively, they demonstrate the versatility of this approach towards almost any type of diblock polysaccharide.

[0122] First, oligoguluronic acid (G) was synthesized using a 1:1 molar ratio between the reactants. n) was investigated. The results for all PDHA-activated oligosaccharides are summarized in Table 2a. The remaining yields were in the range of 40-60%. The preparation of the diblock polysaccharides with reduction and purification is further detailed below.

[0123] [Table 2A]

[0124] The data in Table 2 relate to initial experiments using a 1:1 molar ratio between reactants to obtain reaction kinetics (first order rate constant) and equilibrium yields before further oxime reduction.

[0125] We then synthesized oligoguluronic acids (G n ) was conjugated to an activation block. It is generally found that yields improve when one of the reactants is used in molar excess. In particular, the method for preparation and purification of the diblock may use a molar excess of the activation block relative to the G block.

[0126] For example, yields are significantly improved when oligoguluronic acid (7 mM) is reacted with a three-fold molar excess of PDHA-dextran, reduced and dialyzed. Indeed, our investigations suggest that a 3:1 or 1:3 molar ratio in combination with a subsequent reduction step is necessary to obtain essentially 100% conjugation. When three equivalents of oligoguluronic acid (relative to PDHA-dextran) are used, the diblock could be separated from unreacted oligoguluronic acid by SEC. The best results and the simplest procedure were obtained with three equivalents of PDHA-dextran (relative to oligoguluronic acid), where the diblock could be selectively precipitated with ethanol, while unreacted PDHA-dextran remained in solution and was recycled by standard methods (evaporation / dialysis / lyophilization).

[0127] [Table 2B]

[0128] purification After conjugation, the unreacted G was removed by gel filtration chromatography (GFC) or by acid (added in excess). n The diblock can be purified by either selective precipitation of excess G n Salt or cooling can be used to further promote precipitation of the diblock. Note that conditions should be chosen such that the diblock remains soluble (diblocks with shorter dextran fragments have higher DP). n (which precipitate more easily than those having

[0129] When conjugation is performed with an excess of PDHA-dextran, the pure diblock formed can be selectively precipitated by adding NaCl to a final concentration of 0.2 M, followed by ethanol to 40% (final concentration v / v). The supernatant contains the excess (unreacted) PDHA-dextran, which can be recycled after desalting by dialysis or precipitation with 80% ethanol). Thus, using an excess of the second component is advantageous in terms of both yield and purification.

[0130] Preparation of nanoparticles (NPs) by dialysis or internal gelation: In further embodiments, nanoparticles can be prepared by dialysis or internal gelation (with CaEGTA or CaCO3 / GDL). These two methods result in slightly different particle sizes and also have different assembly kinetics.

[0131] For these examples, the same principles as described above were used to prepare G24-linker-Dex36 diblock polymers.

[0132] Preparation of NPs by internal gelation: 10 mg of G at 22°C 24 -PDHA-Dex 36It was dissolved in 1 ml of 15 mM NaCl and shaken for 12 h. 0.3 ml of 100 mM CaEGTA was added and the solution was filtered (0.22 μm). 0.0166 g of GDL was dissolved in MQ water, filtered and immediately added to the diblock solution. The solution was left at 22 °C for 12 h. Dynamic light scattering (DLS) with backscattering detection (173°) using a ZetaSizer Nano ZS (Malvern Instruments, UK) (25 °C, λ = 632.8) was used to monitor the formation of nanoparticles at regular time intervals (every 1 - 2 h) (scattering intensity (kilo counts per second, kcps) and intensity distribution).

[0133] Preparation of NPs by dialysis: 10 mg of G 24 -PDHA-Dex 36 was dissolved in 1 ml of 10 mM NaCl and shaken for 12 h. The solution was filtered (0.22 μm) and transferred to a dialysis bag. Dialysis against 1 L of 20 mM CaCl2 and 10 mM NaCl 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. Dynamic light scattering (DLS) with backscattering detection (173°) using a ZetaSizer Nano ZS (Malvern Instruments, UK) (25 °C, λ = 632.8) was used to monitor the formation of nanoparticles.

[0134]

Chemical formula

[0135] Stability Dynamic light scattering (DLS) demonstrated the stability of the nanoparticles to a set of different solvent conditions. The nanoparticles were shown to be stable with removal of GDL / EGTA, excess ions (by dialysis against water), and under physiological salt conditions (150 mM NaCl, 1.2 mM CaCl2). The particles were lyophilizable, requiring only heat treatment (40C, 30 min) upon resuspension. The results are presented in Figure 6.

[0136] Using acidification, nanoparticles of G24-b-Dex36 were prepared. Any remaining pure Gn precipitated at low pH, whereas the diblock polymer remained in solution and retained a size corresponding to the nanoparticles. Figure 7 shows this by DLS (dynamic light scattering) analysis presented as a number distribution for various pH values ​​up to 1.09.

[0137] Mg 2+ Stability of nanoparticles in solution G 40 -b-Dex 50 Diblock (4 mg / ml, V = 1.0 ml) was dialyzed (float-A-lyzer 3.5-5.0 kDa) against 20 mM CaCl2 and 10 mM NaCl for 24 h. It was then dialyzed against water (24 h). This method resulted in NPs and some aggregates with this type of diblock.

[0138] The samples were then dialyzed for 20–24 h against solutions (20 ml) containing increasing concentrations of MgCl2 as follows: 0.014 mM, 0.14 mM, 1.4 mM, 14 mM, 140 mM, and 1000 mM. Changes in particle size distribution were monitored by DLS. Ca in the dialysate was measured by ICP-MS. 2+ and Mg 2+ Determine the amount of bound Ca 2+ (X Ca ) and Mg 2+ (X Mg ) fraction was calculated.

[0139] [Table 3]

[0140] This result indicates that bound Ca 2+ Mg 2+ It shows that nanoparticles (nan) remain intact and tend to shrink in size when gradually replaced by ions. Sample 4 (14 mM Mg 2+ , X Ca = 0.77), the smallest particles and narrowest size distribution were obtained. 2+ The concentration of Mg resulted in the expansion of the particles. 2+ Dialysis against salts resulted in the release of strongly bound Ca without particle collapse. 2+ A portion of the ions can be removed.

[0141] Diblock Polymer By reacting free G12 with purified PDHA-dextran bearing DPn100, G 12 -PDHA-Dex 100 A diblock was prepared, where 3 equivalents of G12 were chosen to obtain quantitative substitution of PDHA-dextran. Residual (unreacted) G12 was selectively removed by SEC (Figure 5a). SEC-MALLS data for the diblock showed a clear shift in the elution profile compared to the free block (Figure 5b).

[0142] Nanoparticles Containing Peptide Ligands DP n A polydisperse G block with a .DELTA.=22 was coupled to aminoxy-PEG5 containing a terminal azide group by reductive amination. n -aminooxy-PEG-N3 was further reacted with cyclooctyne (DBCO)-substituted GRGDSP peptide using Cu-free click chemistry to give G n -aminooxy-PEG-peptide was formed.

[0143] By SEC-MALLS, G 25The molar mass of the -aminooxy-PEG-peptide was determined to be 7.9 kDa. Its preparation is described by Solberg et al. (2022) in Carbohydr. Polym. 278, 118840.

[0144] By the GDL / CaEGTA method (20 mM CaEGTA, 3.1 equivalents of GDL), 10% (w / w) G 22 -aminoxy-PEG-peptide and 90% (w / w) G 40 -b-Dex 50 Nanoparticles containing the diblock were prepared with a total diblock concentration of 4 mg / ml.

[0145] This mixture exhibits a G n The nanoparticles formed similarly to the composition without the -aminooxy-PEG-peptide. No free chains (that were not incorporated into nanoparticles) could be detected by DLS after the addition of 0.5 mM BaCl2, which precipitates the free chains. Thus, the normal G n -b-Dex m G to Ziblock n Nanoparticles containing peptide ligands can be prepared by adding -aminoxy-PEG-peptides.

Claims

1. A diblock polymer comprising a first component covalently bonded to a second component via a linker, the first component is an oligomer comprising at least 50 mol % of L-guluronic acid residues and having a degree of polymerization n, where n is at least 3; the second component is a polymer having 30 mol % or less of L-guluronic acid residues and a degree of polymerization m; A diblock polymer where n>=m>=n / 2.

2. A diblock polymer comprising a first component covalently bonded to a second component via a linker, the first component is an oligomer comprising at least 50 mol % of L-guluronic acid residues and having a degree of polymerization n, where n is at least 3; the second component is an oligosaccharide or polysaccharide having 30 mol % or less of L-guluronic acid residues and a degree of polymerization m; A diblock polymer wherein n=>m=>n / 2 and when n is 20 or less, m is 20 or more.

3. A diblock polymer comprising a first component covalently bonded to a second component via a linker, the first component is an oligomer containing at least 50 mol % L-guluronic acid residues; the second component is a second polymer having 30 mol % or less of L-guluronic acid residues; A diblock polymer, wherein the diblock polymer spontaneously forms nanoparticles in an aqueous solution containing metal ions at a metal ion concentration of at least 0.1 mM.

4. The diblock polymer of claim 3, wherein the metal ions comprise Ac, Y, Lu, Cu, Ca, Sr, Ba, or Ra ions, or mixtures thereof.

5. 4. The diblock polymer according to claim 1, wherein the second polymer is an oligo- or polysaccharide, a poly(meth)acrylate, or a polyalkylene glycol, in particular an oligo- or polysaccharide.

6. The diblock polymer according to any one of claims 1 to 3, wherein the second polymer is dextran or pullulan.

7. 4. The diblock polymer according to claim 1, wherein the degree of polymerization n of the L-guluronic acid oligomer is 7 to 70.

8. The diblock polymer according to any one of claims 1 to 3, wherein the degree of polymerization of the second polymer is 8 to 180.

9. The diblock polymer according to any one of claims 1 to 3, wherein the linker is obtained as a result of amination, reductive amination, or click chemistry.

10. The linker may be a triazole, two NH-NH-CO- functional groups, or two -N-O-CH ２ The diblock polymer according to any one of claims 1 to 3, which comprises a -functional group.

11. Nanoparticles comprising the diblock polymer according to any one of claims 1 to 3 and a cation.

12. The nanoparticle of claim 11, wherein the cation is a metal ion.

13. 4. A core-shell nanoparticle comprising the diblock polymer of claim 1, wherein the first component forms the core of the nanoparticle and the second component forms the shell of the nanoparticle; Core-shell nanoparticles in which metal ions and / or charged organic compounds are ionically bound within the core of the nanoparticle.

14. 13. The nanoparticle of claim 12, wherein the metal ion is a Group (II) metal ion or a radionuclide.

15. 12. The nanoparticle of claim 11, further comprising a polymer, including an oligomer, comprising at least 50 mol % of L-guluronic acid residues and having a degree of polymerization n, where n is at least 3, linked to a biologically active molecule.

16. 1. A method for preparing nanoparticles, comprising: (I) obtaining guluronic acid oligomers, such as by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a second polymer having a linking group adapted to react with the guluronic acid oligomer to form a diblock polymer. Contains or or (I) obtaining guluronic acid oligomers, such as by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers; (II) reacting the guluronic acid oligomer with a linking group adapted to react with the guluronic acid oligomer and a second polymer; (III) reacting the guluronic acid oligomer having a linking group with a second polymer to form a diblock polymer; or (I) obtaining guluronic acid oligomers, such as by hydrolyzing alginic acid in the presence of an acid or a base to form guluronic acid oligomers, and further activating the oligomers with functional groups; (II) reacting the guluronic acid oligomer with a second polymer adapted to have a functional group reactive with the functional group of the guluronic acid oligomer to form a diblock polymer; Including, And then, contacting the diblock polymer with a cation, such as a metal ion, a proton, or a charged organic compound, to form nanoparticles.

17. 17. The method of claim 16, wherein the nanoparticles are formed via dialysis or exposure of the nanoparticles to a homogeneous source of metal ions, such as a solution of metal ions.

18. 18. The method of claim 17, wherein exposing the nanoparticles to a homogeneous source of metal ions involves changing the pH of an aqueous solution of the diblock polymer and cations.

19. 17. The method of claim 16, wherein the nanoparticles are contacted with a plurality of second metal ions different from those used in the previous step, such that the plurality of second metal ions at least partially replace the metal ions present in the nanoparticles.

20. 17. The method of claim 16, further comprising contacting the diblock polymer with a cation, e.g., a metal ion, a proton, or a charged organic compound, in the presence of a polymer comprising an oligomer comprising at least 50 mol % L-guluronic acid residues and having a degree of polymerization n, where n is at least 3, linked to a biologically active molecule, e.g., a peptide, to form nanoparticles.

21. 12. Use of the nanoparticles of claim 11 for delivering metal ions or charged organic compounds to a patient or for removing said metal ions from a medium containing said metal ions.

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