Chelating amphiphilic polymers

Abstract

Described are amphiphilic polymers that are provided with chelating moieties. The amphiphilic polymers are block copolymers comprising a hydrophilic block and a hydrophobic block, with the chelating moieties linked to the end-group of the hydrophilic block. The disclosed polymers are capable of self-assembly into structures such as micelles and polymersomes. With suitable metals present in the form of coordination complexes with the chelating moieties, the chelating amphiphilic polymers of the invention are suitable for use in various imaging techniques requiring metal labeling, such as MRI (T.sub.1/T.sub.2 weighted contrast agents or CEST contrast agents) SPECT, PET or Spectral CT.

Claims

1. A particle comprising: a non-chelating amphiphilic polymer; and a self-assembled structure, the self-assembled structure comprising a chelating amphiphilic polymer, wherein the chelating amphiphilic polymer is capable of self-assembly, wherein the chelating amphiphilic polymer comprises a hydrophilic block and a hydrophobic block, wherein the hydrophilic block is provided with a chelating moiety, wherein the chelating moiety is selected from the group consisting of DOTA, DTPA, HYNIC, and desferoxamine as an end-group wherein the hydrophilic block is a poly (ethylene oxide) block, preferably having a weight-average molecular weight of from 500 to 10,000, wherein the hydrophobic block has a Tg of below 70° C., wherein the hydrophobic block is selected from the group consisting of poly (butadiene), poly (isoprene), and poly (ethylethylene).

2. The particle according to claim 1, wherein the non-chelating amphiphilic polymer comprises a poly (oxy ethylene) chain as a hydrophilic block, wherein the chain is longer than the hydophilic block of the chelating amphiphilic polymer.

3. The particle according to claim 1, further comprising a drug.

4. A CEST MRI contrast agent comprising a particle in accordance with claim 1, wherein the particle is a polymersome, wherein the polymersome has a wall formed by a bilayer of one or more amphiphilic polymers, wherein the bilayer comprises a chelating amphiphilic polymer, wherein the wall encloses a cavity, wherein the cavity comprises a pool of proton analytes, wherein the proton analytes are capable of diffusion through the wall, wherein chelating moieties of the chelating amphiphilic polymers extending into the direction of the cavity comprise a chelated paramagnetic material.

5. The CEST MRI contrast agent according to claim 4, wherein the CEST MRI contract agent has a non-spherical shape.

6. A SPECT or PET contrast agent comprising a particle in accordance with claim 1, Wherein the particle is a polymer-stabilized oil-in-water emulsion, Wherein chelating moieties of the chelating amphiphilic polymer are provided with a chelated radionuclide suitable for SPECT or PET.

7. A spectral CT contrast agent comprising a particle in accordance with claim 1, wherein chelating moieties of the chelating amphiphilic polymer are provided with a high-Z material.

8. A method of making a particle, wherein the particle comprises a non-chelating amphiphilic polymer and a self-assembled structure, the self-assembled structure comprising a chelating amphiphilic polymer, wherein the chelating amphiphilic polymer is capable of self-assembly, wherein the chelating amphiphilic polymer comprises a hydrophilic block and a hydrophobic block, wherein the hydrophilic block is provided with a chelating moiety, wherein the chelating moiety is selected from the group consisting of DOTA, DTPA, HYNIC, and desferoxamine as an end-group, wherein one or more of the chelating moieties comprises a metal ion in the form of a coordination complex, wherein the metal ions are chelated at the inside, wherein a chelating amphiphilic polymer is subject to an aqueous environment so as to form a bilayer enclosing a cavity, wherein the chelating moieties are subjected to the formation of coordination complexes with metal ions prior to the formation of the bilayer, the method comprising: creating the amphiphilic polymer; and after creating the amphiphilic polymer coupling the chelating moiety to the hydrophilic block, wherein the hydrophilic block is a poly (ethylene oxide) block, preferably having a weight-average molecular weight of from 500 to 10,000, wherein the hydrophobic block has a Tg of below 70° C., wherein, the hydrophobic block is selected from the group consisting of poly (butadiene), poly (isoprene), and poly (ethylethylene).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. As the color drawings are being filed electronically via EFS-Web, only one set of the drawings is submitted.

(2) FIG. 1 Self-assembled nanostructures containing amphiphilic copolymers. Polymersomes (left), polymer-stabilized emulsions (middle), and polymeric micelles (right).

(3) FIG. 2 Schematic representation of Gd(III)DOTA-functionalized polymersomes as T.sub.1,2-weighted contrast agents for Magnetic Resonance Imaging (MRI).

(4) FIG. 3 Schematic representation of DOTA-functionalized polymersomes. Spherical polymersomes containing DOTA-terminated copolymers in the polymer layer (6, left). The reaction of the DOTA-moieties with paramagnetic metals yields spherical polymersomes (10, middle), in which the paramagnetic complexes point outward to the bulk water. Deformation of the polymersomes (10) in response to osmotic pressure affords aspherical polymersomes (11, right).

(5) FIG. 4 Schematic representation of DOTA-functionalized polymersomes containing a chemical shift agent in the inner aqueous compartment. Spherical polymersomes containing DOTA-terminated copolymers in the polymer layer (12, top left). The reaction of the DOTA-moieties of 12 with a paramagnetic metal yields spherical polymersomes (13, top middle), in which the paramagnetic complexes point outward to the bulk water. The deformation of 12 in response to osmotic pressure gives aspherical polymersomes (15, bottom). Aspherical polymersomes containing a chemical shift agent in the inside and paramagnetic complexes pointing outward to the bulk water (14, top right) can be obtained from either 13 or 15.

(6) FIG. 5 Schematic representation of polymersomal CEST MRI contrast agents containing paramagnetic complexes of DOTA copolymers on both sides of the polymer layer. Spherical polymersomes (16, top left), aspherical polymersomes (17, top right), spherical polymersomes containing a chemical shift agent in the inner aqueous compartment (18, bottom left), and aspherical polymersomes containing a chemical shift agent in the inner aqueous compartment (19, bottom right).

(7) FIG. 6 Polymersomes, polymer-stabilized emulsions, and polymeric micelles for nuclear imaging, showing at the outside of the structure a label e.g. for nuclear imaging.

(8) FIG. 7 SPECT/CT image of .sup.111Indium-labeled emulsion 4 hours post-injection. Maximum intensity projection of CT coregistrated with the SPECT image (top left); coronal SPECT/CT slice visualizing the heart and liver (top right); sagittal SPECT/CT slice visualizing the heart, liver and kidney (bottom left); transversal SPECT/CT slice (bottom right).

EXAMPLES

Example 1

Synthesis of DOTA-Functionalized poly(ethylene oxide)-block-poly(butadiene) (5)

(9) PBD(2500)-b-PEO(1300) (1) was dissolved in acetone (18 mL) and the solution was concentrated under reduced pressure in order to remove residual isopropanol. In order to remove traces of water, the copolymer was dissolved in toluene (15 mL) and this solution was concentrated in vacuo. Subsequently, PBD(2500)-b-PEO(1300) (4.90 g, 1.29 mmol) was dissolved in DCM (15 mL) under an atmosphere of nitrogen. The obtained solution was cooled till 0° C. and p-tosylchloride (0.497 g, 2.60 mmol) was added. The mixture was stirred for 30 min at 0° C. and KOH (0.640 g, 11.4 mmol) was added gently. The mixture was stirred overnight at room temperature. The reaction mixture was washed with water (2×30 mL) and brine (2×15 mL). The aqueous layer was extracted with DCM (30 mL) and the combined organic layers were dried over MgSO.sub.4, filtered and the solution was concentrated under reduced pressure to yield 2 (62%, 3.2 g, 0.81 mmol). The tosylate-functionalized copolymer (2) (3.2 g, 0.81 mmol) was dissolved in toluene (12 mL) and a solution of 7 N NH.sub.3 in MeOH (12 mL, 84 mmol) was added. The reaction was performed at 50° C. for 63 h. Then, the solvent was removed under reduced pressure. The crude mixture was dissolved in DCM (10 mL). The obtained solution was washed with water (2×20 mL), brine (2×10 mL), and saturated NaHCO.sub.3 (aq) (10 mL). The aqueous layer was extracted with DCM (40 mL). The combined organic layers were dried over MgSO.sub.4. The suspension was filtered and the filtrate was concentrated under reduced pressures to give 3 (1.55 g, 0.41 mmol) in a yield of 50%. The amine-functionalized copolymer (3) (1.2 g, 0.31 mmol) was dissolved in DME (12 mL) and, subsequently, DOTA-based building block (4) (0.347 g, 0.35 mmol) and Et.sub.3N (0.9 mL, 6.5 mmol) were added. The mixture was stirred for 26 hrs at room temperature under an atmosphere of nitrogen. The obtained solution was concentrated under reduced pressure. The crude mixture was dissolved in toluene and the solution was concentrated under reduced pressure. The DOTA-functionalized poly(ethylene oxide)-block-poly(butadiene) (5) was obtained in quantitative yield.

Example 2

(10) Self-Assembly of DOTA-Functionalized Copolymers and the Complexation with Gd(III)

(11) Polymer vesicles with an average diameter of 100-150 nm were formed by the thin film hydration technique coupled with sequential extrusions. In brief, the DOTA-functionalized poly(butadiene(1,2-addition)-b-ethylene oxide) (M.sub.n(g/mol): PBD(2500)-b-PEO(1300), PD=1.04, and f.sub.EO=0.34) was dissolved in CHCl.sub.3. The solvent was gently removed under reduced pressure and a thin polymer film was obtained. The film was hydrated in 20 mM HEPES solution (pH 7.4). After overnight heating at 50° C. followed by ten freeze-thaw cycles at −177° C. and 70° C., the dispersion was extruded several times through polycarbonate filters with a pore diameter of 1 μm, 0.4 μm, 0.2 μm, and 0.1 μm. Subsequently, a solution of GdCl.sub.3 (5 equivalents) in 20 mM HEPES solution at pH 7.4 was added to the polymersome dispersion at 50° C. for 2 hours. Subsequently, the polymersomes were dialyzed overnight to remove the excess of Gd(III). Dialysis was performed against a 20 mM HEPES solution at pH 7.4. The mean average radius of the polymersomes was determined by dynamic light scattering (DLS). The shape of the polymer vesicles was studied by cryo-TEM. The concentration of gadolinium was determined by ICP-MS. The longitudinal and transverse relaxation times (T.sub.1 and T.sub.2) were determined at 60 MHz.

Example 3

(12) P Aspherical Polymersomes Containing a Chemical Shift Agent and Paramagnetic Complexes of DOTA-Terminated Polymers (14)

(13) Polymer vesicles with an average diameter of 100-150 nm were formed by the thin film hydration technique coupled with sequential extrusions, as described in example 1. In this case the film was hydrated in 20 mM HEPES solution (pH 7.4) containing 65 mM [Tm(hpdo3a)(H.sub.2O)]. After overnight heating at 50° C. followed by ten freeze-thaw cycles at −177° C. and 70° C., the dispersion was extruded several times through polycarbonate filters with a pore diameter of 1 μm, 0.4 μm, 0.2 μm, and 0.1 μm. The obtained polymersomes (12) were dialyzed overnight to remove [Tm(hpdo3a)(H.sub.2O)] that was not entrapped after hydration of the lipidic film, and to obtain aspherical polymersomes (15). Dialysis was performed with a 20 mM HEPES buffer containing 0.3 M NaCl. Subsequently, a solution of TmCl.sub.3 (5 equivalents) in 20 mM HEPES buffer containing 0.3 M NaCl was added to the polymersome dispersion at 50° C. for 2 hours. The polymersomes (14) were dialyzed overnight to remove the excess of Tm. Dialysis was performed against a 20 mM HEPES buffer containing 0.3 M NaCl (pH 7.4). The mean average radius of the polymersomes (14) was determined by dynamic light scattering (DLS). The shape of the polymer vesicles was studied by cryo-TEM. The concentration of gadolinium was determined by ICP-MS. The longitudinal and transverse relaxation times (T.sub.1 and T.sub.2) were determined at 60 MHz.

Example 4

(14) Radiolabeled Polymersomes and Emulsions

(15) Preparation of the Polymer-Stabilized Emulsion

(16) Emulsions were prepared from octan-2-yl 2,3,5-triiodobenzoate (25% weight/volume) using 2% weight/weight of poly(butadiene(1,2 addition)-block-poly(ethylene oxide) (f.sub.EO 0.61; Mw.sub.phil=2033 g/mol; Mw.sub.phob=1305 g/mol) and 5 mol % of DOTA-functionalized copolymer (5). The emulsions were prepared in a 2.1 mM THAM buffer containing 152 mM NaCl at pH 7.4 using a high pressure microfluidizer system (Microfluidizer M110S, Microfluidics Int. Corp., Newton Mass.) at 70° C. Extensive dialysis was performed against a THAM buffer (1 L) containing Chelex (2 g/L) for three days. Subsequently, the polymer-stabilized emulsions were filtrated through a 450 nm filter.

(17) Radiolabeling of the Emulsions

(18) The emulsion stabilized with the DOTA-copolymer (300 μL) was incubated with 30 MBq of .sup.111InCl.sub.3 in 0.05 M HCl (4 μL).sup.1 for 1 hour at 70° C. Subsequently, free DTPA was added to the reaction mixture to scavenge free .sup.111In. 1 μL of the reaction mixture was applied on a silica-coated TLC plate. A solution of 200 mM EDTA containing 9.0 g/L NaCl was used as an eluent. The TLC was analyzed on a FLA-7000 phosphoimager (Fuji Film, Tokyo, Japan) and the radiolabeling was quantified using Aida software (Fuji film). The radiolabeling efficiency was 65% with 30 MBq of .sup.111InCl.sub.3. The radiolabeling on a smaller scale (4.6 MBq of .sup.111InCl.sub.3 in 100 μL emulsion) gave a yield of 97%. Although this yield is higher, 4.6 MBq would not be enough for imaging purposes. Therefore, the described procedure with 30 MBq .sup.111In was used for in-vivo studies. The radiolabeled emulsions were tested in male Swiss mice (Charles River, Maastricht, the Netherlands) for a dual modality SPECT/CT scan. The radiolabeled emulsion (200 μL) with an activity of 20.5 MBq was injected intravenously. SPECT/CT scans were performed on a NanoSPECT/CT (Bioscan). The animal experiment was approved by the Institutional Ethical Review Committee for animal experiments of the Maastricht University (Maastricht, the Netherlands).