DIBLOCK POLYMER

Abstract

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 and having a degree of polymerisation n where n is at least 3; said second component is a polymer having no more than 30 mol % L-guluronic acid residues and having a degree of polymerisation m; wherein 9n>=m>=n/2.

Claims

1. 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 and having a degree of polymerisation n where n is at least 3; said second component is a polymer having no more than 30 mol % L-guluronic acid residues and having a degree of polymerisation m; wherein 9n>=m>=n/2.

2. 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 and having a degree of polymerisation n where n is at least 3; said second component is an oligo or polysaccharide having no more than 30 mol % L-guluronic acid residues and having a degree of polymerisation m; wherein 9n=>m=>n/2 and wherein m is 20 or more if n is 20 or less.

3. 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; said second component is a second polymer having no more than 30 mol % L-guluronic acid residues; wherein said diblock polymer forms a nanoparticle spontaneously in an aqueous solution comprising metal ions in a concentration of at least 0.1 mM of metal ions, such as Ac, Y, Lu, Cu, Ca, Sr, Ba or Ra ions or mixtures thereof, especially Ca, Sr, Ba or Ra ions.

4. A diblock polymer 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 diblock polymer as claimed in any preceding claim wherein the second polymer is a dextran or pullulan.

6. A diblock polymer as claimed in any preceding claim wherein the L-guluronic acid oligomer has a degree of polymerisation n of 7 to 70.

7. A diblock polymer as claimed in any preceding claim wherein the second polymer has a degree of polymerisation of 8 to 180.

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

9. A diblock polymer as claimed in any preceding claim wherein the linker comprises a triazole, two NHNHCO functional groups or two NOCH.sub.2 functional groups.

10. A nanoparticle comprising a diblock polymer as claimed in claims 1 to 9 and positive ions such as metal ions, such as metal 2+ or 3+ ions.

11. A core shell nanoparticle comprising a diblock polymer as claimed in any one of claims 1 to 10, said first component forming the core and said second component forming the shell of said nanoparticle, wherein metal ions and/or charged organic compounds are ionically bound within the core of the nanoparticle.

12. A nanoparticle as claimed in claim 10 or 11 wherein said metal ions are group (II) metal ions or radionuclides.

13. A nanoparticle as claimed in claims 10 to 12 further comprising a polymer comprising an oligomer comprising at least 50 mol % L-guluronic acid residues and having a degree of polymerisation n where n is at least 3 linked to a biological active molecule such as a peptide.

14. A process for the preparation of a nanoparticle comprising: (I) obtaining a guluronic acid oligomer such as by hydrolysing alginate in the presence of an acid or base to form a guluronic acid oligomer; (II) reacting said guluronic acid oligomer with a second polymer carrying a linking group adapted to react with said guluronic acid oligomer to form a diblock polymer. or (I) obtaining a guluronic acid oligomer such as by hydrolysing alginate in the presence of an acid or base to form a guluronic acid oligomer; (II) reacting said guluronic acid oligomer with a linking group adapted to react with said guluronic acid oligomer and with a second polymer; (III) reacting said guluronic acid oligomer with linking group with a second polymer to form a diblock polymer; or (I) obtaining a guluronic acid oligomer, such as by hydrolysing alginate in the presence of an acid or base to form a guluronic acid oligomer, and activating said oligomer with a functional group; (II) reacting said guluronic acid oligomer with a second polymer which is adapted to carry a functional group that reacts with the functional group of the guluronic acid oligomer to form a diblock polymer; and subsequently: contacting said diblock polymer with positive ions, such as metal ions, protons or a charged organic compound to form nanoparticles.

15. A process as claimed in claim 14 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.

16. A process as claimed in claim 15 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.

17. A process as claimed in claims 14 to 16 wherein said nanoparticles are contacted with a plurality of second metal ions different from those used in the previous step so that said plurality of second metal ions at least partially displace said the metal ions present in said nanoparticles.

18. A process as claimed in claims 15 to 17 further comprising contacting said diblock polymer with positive ions, such as metal ions, protons or a charged organic compound to form nanoparticles in the presence of a polymer comprising an oligomer comprising at least 50 mol % L-guluronic acid residues and having a degree of polymerisation n where n is at least 3 linked to a biological active molecule such as a peptide.

19. Use of a nanoparticle as claimed in claims 10 to 13 to deliver a metal ion or charged organic compound to a patient or to remove a metal ion from a medium containing said metal ion.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0141] FIG. 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. FIG. 1 also shows the subsequent dimerization with Ca++ and G.sub.n-L-Dex.sub.m to form particles. The formation of these dimers leads, in turn to the formation of nanoparticles.

[0142] FIG. 2 shows the reaction of guluronate with PDHA or ADH and subsequent reduction using PB.

[0143] FIG. 3 shows the NMR and chemical structure of Dex.sub.10-PDHA=G.sub.3 where n represents the reduced N-oxide. The figure shows the conjugaton prior to reduction with the Schiff base. .sup.1H-NMR spectra of the equilibrium reaction mixture with G.sub.3 and PDHA-Dex.sub.10 is taken 500 mM AcOH[d.sub.4] pD 4. Resonances from (E)/(Z)-oximes of the conjugate are annotated. The structure of the conjugated G.sub.n=b-Dex.sub.m is included (=refers to unreduced oxime). .sup.1H-NMR spectra of purified Dex.sub.10-PDHA is included for comparison.

[0144] FIG. 4 is a theoretical depiction of core shell nanoparticles of the invention with radionuclides coordinated in the core or via antibodies attached to the shell.

[0145] FIG. 5 shows G.sub.12-PDHA-Dex.sub.100 diblock polymer data. Residual (unreacted) G.sub.12 was selectively removed by SEC (FIG. 5a). SEC-MALLS data for the diblock showed a clear shift in elution profile compared to the free blocks (FIG. 5 b).

[0146] FIG. 6 shows that nanoparticles made by G.sub.24-b-Dex.sub.36 remain stable (have the same particle sizes) after various treatments.

[0147] FIG. 7 shows G.sub.24-b-Dex.sub.36 nanoparticle sizes as a function of pH.

EXAMPLES

Test Methods

SEC-MALS

[0148] The molecular weight and intrinsic viscosity of the block polymers (G.sub.n-b-G.sub.n and G.sub.n-b-Dex.sub.m) was analysed by Size Exclusion Chromatograph (SEC) with Multiangle Light Scattering (MALS). Samples were dissolved in the mobile phase (0.15 M NaNO.sub.3 with 10 mM EDTA) and filtered (0.45 m) 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 L per injection volume) by an Agiel Technologies Vialsampler. TKS Gel columns 4000 and 2500 were connected in series. DAWN Heleos-II and ViscoStar II detectors from Wyatt Technology were connected in series with a Shodex refractive index detector (RI-5011). Astra 7.3.0 software was used for data collection and processing.

Preparation of Guluronic Acid Oligomers

[0149] 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 DP.sub.n. DP.sub.n was determined by NMR.

[0150] The following Guluronic acid oligomers are prepared:

[0151] DP 21, F.sub.G 0.90 (where DP.sub.n is the average degree of polymerization and F.sub.G is the fraction of monomers that are guluronic acid, i.e. the mol % of guluronic acid). [0152] DP4. The F.sub.G of the sample was >0.9. [0153] DP10. The F.sub.G of the sample was >0.9. [0154] DP11. The F.sub.G of the sample was >0.9. [0155] DP12. The F.sub.G of the sample was >0.9.

[0156] The guluronic acid oligomers are then activated to form conjugates or combined with activated dextran components to form a diblock polymer.

[0157] Adipic acid dihydrazide (ADH), O,O-1,3,-propanediylbishydroxylamine dihydrochloride (PDHA) and 2-methylpyridine borane complex (-picoline borane-PB) was purchased from Sigma-Aldrich.

Preparation of Guluronate ConjugatesGeneral Protocol

[0158] 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 DPn7 with 3.5 kDa MWCO) first against 50 mM NaCl, then against MQ water. Excess linker was removed by semi-preparative SEC, after which samples were dialyzed and freeze-dried. FIG. 2 depicts reactions which occur. These conjugates can be combined with the second polymer.

Comparative Preparation of Guluronate Diblocks

[0159] 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.

Inventive Preparation of Guluronate-Linker-dextran Block CopolymersGeneral Protocol

[0160] 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 semi-preparative GFC, dialysis and freeze drying.

Particle Formation of G-Linker-Dextran

[0161] G.sub.n-Linker-Dex.sub.m (n=12 and m=100) (5-10 mg/ml) was dissolved in 1 ml 10 mM NaCl and filtered (0.22 m). After 24 h, the sample was dialyzed (Float-A-Lyzer 100-500 Da) against 20 mM CaCl.sub.2 with 10 mM NaCl (1-1.5 L).

[0162] The Mn, Mw, and DP.sub.n from SEC MALS analyses of Dex.sub.m-b-G.sub.n block copolymer (after purification by SEC) and the starting material (G.sub.n and Dex.sub.m-Linker) is presented in table 1.

TABLE-US-00001 TABLE 1 Sample M.sub.n (kDa) M.sub.w (kDa) DP.sub.n G.sub.n 2.5 2.5 12 Dex.sub.m-Linker 16.2 18.3 100 Dex.sub.m-Linker-G.sub.n 18.3 20.3 112

[0163] For proof of concept, a further diblock polymer was prepared following the same protocols above and was analysed using NMR. In order to make the NMR easier to assign, shorter chain dextran and guluronic acid oligomers were used. FIG. 3 is the NMR spectra for the Dex.sub.10-PDHA-G.sub.3 of the equilibrium reaction mixture with G.sub.3 and PDHA-Dex.sub.10 (1:1) in 500 mM AcOHd4 pD 4 (600 MHZ). Resonances from (E)/(Z)-oximes of the conjugate are annotated. The structure of the conjugated G.sub.3=b-Dex.sub.m is included (=refers to unreduced oxime). 1H-NMR spectra of purified Dex.sub.10-PDHA is included for comparison.

[0164] In conclusion, the conjugation of oligoguluronate with PDHA-activated dextran chains is efficient for longer and shorter chains, as demonstrated with a DP.sub.100 dextran chain and a DP.sub.10 dextran chain.

Block Copolymer Self-Assembly in Solution

[0165] G.sub.40-linker-Dex.sub.100 diblock polymer in solution was combined with CaCl.sub.2 (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 G.sub.40 blocks alone precipitate under similar conditions. Therefore, the diblock structure enabled a strict phase separation between the G-based core and the dextran corona.

Block Copolymer Self-Assembly in Solution

[0166] G.sub.11-b-Dex.sub.100 was prepared analogously. G.sub.11-b-Dex.sub.100 has a markedly different behaviour under similar conditions. Namely, the block copolymer tended to form larger nanoparticles in solution with Ca (1000 nm or more). From a thermodynamic point of view, this could mean that the loss of entropy associated with the formation of a dextran corona is not compensated by a sufficient gain in enthalpy through the gelling of G blocks as they are shorter. Therefore, the ratio of the two blocks length must be carefully considered to have self-assembly properties.

Further Diblock Polymers

[0167] The high reactivity of oligouronates with PDHA implies that reaction with PDHA-activated oligosaccharides to obtain diblock oligo-or polysaccharides would proceed with similar results. This was tested in kinetic studies with -1,3-glucan-PDHA (DP.sub.9).

[0168] In addition, the reaction was also studied with G.sub.n-PDHA for preparation of symmetrical blocks. All conjugates (oximes) had been fully reduced with picoline borane (PB) prior to coupling with G.sub.3. These PDHA-activated oligosaccharides represent widely different chemistries (Table 3): dextrans are neutral chains with high chain flexibility due to -1,6 linkages. Amylose (-1,4-linked glucans) and -1,3-glucans are both semi-rigid, neutral chains with the ability to form higher order structures. Collectively they illustrate the versatility of the approach towards almost any type of diblock polysaccharides.

[0169] The conjugations with oligoguluronates (G.sub.n) were initially studied using a 1:1 molar ratio between the reactants. Results for all PDHA-activated oligosaccharides are summarised in table 2a. Yields were otherwise in the range 40-60%. The preparation of diblock polysaccharides with reduction and purification is further detailed below.

TABLE-US-00002 TABLE 2a Coupling of PDHA-activated oligosaccharides to oligoguluronates (G.sub.3). Second Equ. yield (%) Activated block block Ratio Conc. B prior to oxime (A*) (B) (A*-B) [mM] pH reduction G.sub.10-PDHA.sup.# G.sub.3 1:1 7.0 4 45 Dex.sub.10-PDHA G.sub.3 1:1 7.0 4 42 -1,3-glucan-PDHA G.sub.3 1:1 7.0 4 61 .sup.#comparative example

[0170] The data in table 2 concerns initial experiments using a 1:1 molar ratio between the reactants to obtain reaction kinetics (first order rate constants) and equilibrium yields prior to further oxime reduction.

[0171] We subsequently conjugated oligoguluronates (G.sub.n) to the activated block using molar ratios in which one or other of the reactants was in molar excess. We generally find that yields are improved where a molar excess of one of the reactants is employed. In particular, the method for diblock preparation and purification might use a molar excess of the activated block relative to the G-block.

[0172] For example when the oligoguluronate (7 mM) is reacted with a 3-fold molar excess of PDHA-dextran, reduced, and dialysed, yields are markedly improved. Our research suggests in fact that a 3:1 or 1:3 molar ratio combined with a subsequent reduction step was needed to obtain essentially 100% coupling. If three equivalents of oligoguluronate (relative to PDHA-dextran) were used the diblock could be separated from unreacted oligoguluronate by SEC. Best results and simplest procedures were obtained with three equivalents of PDHA-dextran (relative to oligoguluronate), where the diblock could be selectively precipitated with ethanol while unreacted PDHA-dextran remained in solution and was recycled by standard methods (evaporation/dialysis/freeze-drying).

TABLE-US-00003 TABLE 2b Coupling of PDHA-activated oligosaccharides to oligoguluronates (G.sub.3). Second Yield after Activated block block Ratio Conc. B oxime (A*) (B) (A*-B) [mM] pH reduction (%) Dex45-PDHA G19 3:1 7.0 4 100

Purification

[0173] After coupling, the diblocks can be purified either by gel filtration chromatography (GFC) or by selective precipitation of unreacted G.sub.n (added in excess) with acid. Salt or cooling can be used to further drive the precipitation of excess G.sub.n. Noticeably, the conditions should be chosen so that the diblock remains soluble (diblocks short dextran will precipitate more easily compared to one with a higher DP.sub.n).

[0174] When coupling is carried out with an excess of PDHA-dextran, the pure diblock that is 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). There are therefore advantages to the use of excess of the second component both in terms of yield and purification.

Preparation of Nanoparticles (NPs) by Dialysis or Internal Gelation:

[0175] In a further embodiment, nanoparticles can be prepared by dialysis or internal gelation (with CaEGTA or CaCO.sub.3/GDL). The two methods give slightly different particles size and also have different kinetics of assembly.

[0176] For these examples a G24-linker-Dex36 diblock polymer was prepared using similar principles to those described above.

Preparation of NPs by Internal Gelation:

[0177] 10 mg G.sub.24-PDHA-Dex.sub.36 was dissolved in 1 ml 15 mM NaCl 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 m). 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., =632.8) with back scattering detection (173).

Preparation of NPs by Dialysis:

[0178] 10 mg G.sub.24-PDHA-Dex.sub.36 was dissolved in 1 ml 10 mM NaCl at 22 C. and placed on shaking for 12 h. The solution was filtered (0.22 m) and transferred to a dialysis bag. Dialysis against 1 L 20 mM CaCl.sub.2 with 10 mM NaCl was continued for 20 h for MWCO3.5 kDa, 14 days for 0.5 kDa<MWCO1.0 kDa and 14 days for MWCO0.5 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., =632.8) with back scattering detection (173).

[0179] Scheme 1 shows the reactions which occur:

Stability

[0180] The stability of the nanoparticles for a set of different solvent conditions was demonstrated by dynamic light scattering (DLS). The nanoparticles were shown to be stable upon removal of GDL/EGTA, excess ions (by dialysis against water), and under physiological salt conditions (150 mM NaCl, 1.2 mM CaCl2). The particles could be freeze dried (upon resuspension only a heat treatment (40 C, 30 min) is needed). Results are presented in FIG. 6.

[0181] Nanoparticles of G24-b-Dex36 were prepared using acidification. Any residual pure Gn precipitates at low pH, whereas the diblock polymer remains in solution and retains a size corresponding to nanoparticles. The FIG. 7 show this by DLS (dynamic light scattering) analysis presented as number distributions for various pH values down 1.09.

Nanoparticle Stability in Mg.SUP.2+ .Solutions.

[0182] A G.sub.40-b-Dex.sub.50 diblock (4 mg/ml, V=1.0 ml) was dialyzed (float-A-lyzer 3.5-5.0 kDa) against 20 mM CaCl.sub.2 with 10 mM NaCl for 24 h. It was then dialyzed against water (24 h). The process gave NPs and some aggregates with this type of diblock.

[0183] The sample was subsequently dialysed for 20-24 h against solutions (20 ml) containing stepwise increasing concentrations of MgCl.sub.2: 0.014 mM, 0.14 mM, 1.4 mM, 14 mM, 140 mM and 1000 mM. The changes in particle size distribution were monitored by DLS. The amounts of Ca.sup.2+ and Mg.sup.2+ ions in the dialysate were determined by ICP-MS from which the fractions of bound Ca.sup.2+ (X.sub.Ca) and Mg.sup.2+ (X.sub.Mg) were calculated.

TABLE-US-00004 TABLE 3 Mg.sup.2+ dialysis Fraction of bound Ca.sup.2+ Sample no solution (mM) (X.sub.Ca) 1 0.014 1.0 2 0.14 0.87 3 1.4 0.92 4 14 0.77 5 140 0.65

[0184] The results show that the nan remain intact and tend to shrink in size when bound Ca.sup.2+ is gradually replaced by Mg.sup.2+ ions. The smallest particles and the narrowest size distributions were obtained for sample 4 (14 mM Mg.sup.2+, X.sub.Ca=0.77). Higher Mg.sup.2+ concentrations led to particle swelling. Dialysis against appropriate concentrations of Mg.sup.2+ salts can therefore remove some of the strongly bound Ca.sup.2+ ions without particle disintegration.

Diblock Polymer

[0185] G.sub.12-PDHA-Dex.sub.100 diblock was prepared by reacting free G12 with purified PDHA-dextran with DPn 100. Three equivalents of G12 were here chosen to obtain quantitative substitution of the PDHA-dextran. Residual (unreacted) G12 was selectively removed by SEC (FIG. 5a). SEC-MALLS data for the diblock showed a clear shift in elution profile compared to the free blocks (FIG. 5 b).

Nanoparticles Containing a Peptide Ligand

[0186] A polydisperse G-block with DP.sub.n=22 was coupled to aminoxy-PEG5 containing a terminal azide group by reductive amination. The G.sub.n-aminooxy-PEG-Na was further reacted with cyclooctyne (DBCO) substituted GRGDSP peptide using Cu-free click chemistry to form the G.sub.n-aminooxy-PEG-peptide.

[0187] The molar mass of the G25-aminooxy-PEG-peptide of 7.9 kDa was determined by SEC-MALLS. The preparation is described in Solberg et al (2022) Carbohydr. Polym. 278, 118840.

[0188] Nanoparticles containing 10% (w/w) of G.sub.22-aminoxy-PEG-peptide and 90% (w/w) of a G.sub.40-b-Dex.sub.50 were prepared by the GDL/CaGEGTA method (20 mM CaEGTA, 3.1 equivalents of GDL). The total diblock concentration was 4 mg/ml.

[0189] The mixture forms nanoparticles similarly to compositions without the G.sub.n-aminooxy-PEG-peptide with only slightly higher hydrodynamic values. No free chains (not incorporated into nanoparticles) could be detected by DLS after adding 0.5 mM BaCl.sub.2, which precipitates free chains. Hence, nanoparticles containing a peptide ligand can be prepared by adding G.sub.n-aminoxy-PEG-peptide to a normal G.sub.n-b-Dex.sub.m diblock.