PHOSPHORUS-31 MRI AGENTS

Abstract

Use of a P-containing polymer for measuring .sup.31P-MRI, wherein the P-containing polymer is selected from polyphosphates, polyphosphonates, poly(phosphine oxide)s, polyphosphazenes, polyphosphinates, polyphosphoramidates, polyphosphorodiamidates, polyphosphoamides, polythionophosphates, and polythionophosphonates. A polyphosphonate copolymer and an aqueous suspension comprising micelles of the polyphosphonate copolymer.

Claims

1. A method, comprising the step of: using a P-containing polymer for measuring .sup.31P-MRI, wherein the P-containing polymer is selected from polyphosphates, polyphosphonates, poly(phosphine oxide)s, polyphosphazenes, polyphosphinates, polyphosphoramidates, polyphosphorodiamidates, polyphosphoamides, polythionophosphates, and polythionophosphonates.

2. The method according to claim 1, wherein the amount of P in the polymer is at least 3 wt. %, preferably at least 5 wt. %, more preferably at least 10 wt. %, most preferably at least 15 wt. %.

3. The method according to claim 1, wherein the polymer has a T.sub.g below 37 C., preferably below 35 C., more preferably below 30 C., most preferably below 25 C. or 20 C.

4. The method according to claim 1, wherein the polymer has a T.sub.1 between 0.05 s and 5 s, a T.sub.2 between 0.03 s and 3 s, and wherein T.sub.1>T.sub.2; preferably a T.sub.1 between 0.1 s and 3 s, and a T.sub.2 between 0.04 s and 1.5 s; and more preferably a T.sub.1 between 0.5 s and 2.5 s, and a T.sub.2 between 0.045 and 1.5, wherein T.sub.1 and T.sub.2 are the longitudinal and transverse relaxation times calculated using monoexponential decay.

5. The method according to claim 1, wherein the P-containing polymer is a polyphosphonate.

6. The method according to claim 5, wherein the polyphosphonate is a copolymer.

7. The method according to claim 6, wherein the polyphosphonate copolymer comprises at least two monomeric units A and B, wherein monomeric unit ##STR00013## and monomeric unit ##STR00014## and wherein R.sub.1 and R.sub.3 each independently are ##STR00015## wherein n>1, preferably wherein 1<n<10; or ##STR00016## wherein n>0, preferably wherein 0<n<10; or ##STR00017## wherein n>0, preferably wherein 0<n<10; or ##STR00018## wherein n>0, preferably wherein 0<n<10; and wherein R.sub.2 represents optionally substituted phenyl, optionally substituted benzyl, or optionally substituted phenethyl; and R.sub.4 represents straight or branched C.sub.1-6 alkyl, straight or branched C.sub.2-6 alkenyl, straight or branched C.sub.1-6 alkoxy, or straight or branched C.sub.1-6 alkanoyl.

8. The method according to claim 7, wherein R.sub.1 and R.sub.3 are a) with n=2; R.sub.2 represents methylphenyl, dimethylphenyl, ethylphenyl, methylbenzyl, or phenethyl; and R.sub.4 represents straight or branched C1-4 alkyl, straight or branched C2-4 alkenyl, straight or branched C1-4 alkoxy, or straight or branched C1-4 alkanoyl, preferably wherein R.sub.2 represents phenyl, and R.sub.4 represents ethyl.

9. The method according to claim 7, wherein the polyphosphonate copolymer comprises from 30-70 mol % A, and from 30-70 mol % B, preferably from 35-65 mol % A, and from 35-65 mol % B, most preferably from 40-60 mol % A, and from 40-60 mol % B, the total of A and B adding up to 100 mol %.

10. The method according to claim 7, wherein the polyphosphonate copolymer is a diblock copolymer, a normal tapered block copolymer, or a gradient copolymer, preferably wherein the polyphosphonate copolymer is a gradient copolymer which has a gradient as defined in Gleede et al., Macromolecules 2019, 52 (24), 9703-9714 of block(-like), medium or hard, preferably of medium or hard.

11. The method according to claim 10, wherein the polyphosphonate copolymer is a gradient copolymer which has a value relative to the length of the minority monomeric unit as defined by Shull, Interfacial activity of gradient copolymers, Macromolecules 2002, 35, 8631-8639 of 0.5-1, preferably 0.6-1, more preferably of 0.8-1.

12. The method according to claim 7, wherein the polyphosphonate copolymer has a molecular weight (M.sub.n) between 1000 and 100.000, and a dispersity between 1.01 and 10.00.

13. A polyphosphonate copolymer, which comprises: two monomeric units A and B, wherein monomeric unit ##STR00019## and monomeric unit ##STR00020## and wherein R.sub.1 and R.sub.3 are ##STR00021## wherein n=2-4, and wherein R.sub.2 represents phenyl, benzyl, or phenethyl; R.sub.4 represents methyl, ethyl or propyl; and wherein the copolymer comprises from 40-60 mol % A, and from 60-40 mol % B, and wherein the polyphosphonate copolymer is a diblock copolymer, a normal tapered block copolymer, or a gradient copolymer, preferably wherein the polyphosphonate copolymer is a gradient copolymer; preferably wherein R.sub.1 and R.sub.3 are ##STR00022## wherein n=2, and wherein R.sub.2 represents phenyl; R.sub.4 represents ethyl.

14. The polyphosphonate copolymer according to claim 13, wherein the polymer is a gradient copolymer, wherein r.sub.A is between 4.3 and 24.4, and wherein r.sub.B is between 0.03 and 0.25.

15. An aqueous suspension, comprising: micelles of the polyphosphonate copolymer of claim 13, wherein the micelles have a hydrodynamic radius R.sub.h as defined by ISO 22412:2008 of between 5 and 100 nm, a PDI of 0.001-0.5 as defined by ISO 22412:2008, and a concentration of between 1-500 mg/mL, preferably 5-490 mg/mL, more preferably 10-480 mg/mL.

16. The method according to claim 2, wherein the polymer has a T.sub.g below 37 C., preferably below 35 C., more preferably below 30 C., most preferably below 25 C. or 20 C., wherein the polymer has a T.sub.1 between 0.05 s and 5 s, a T.sub.2 between 0.03 s and 3 s, and wherein T.sub.1>T.sub.2; preferably a T.sub.1 between 0.1 s and 3 s, and a T.sub.2 between 0.04 s and 1.5 s; and more preferably a T.sub.1 between 0.5 s and 2.5 s, and a T.sub.2 between 0.045 and 1.5, wherein T.sub.1 and T.sub.2 are the longitudinal and transverse relaxation times calculated using monoexponential decay, and wherein the P-containing polymer is a polyphosphonate.

17. The method according to claim 16, wherein the polyphosphonate is a copolymer, wherein the polyphosphonate copolymer comprises at least two monomeric units A and B, wherein monomeric unit ##STR00023## and monomeric unit ##STR00024## and wherein R.sub.1 and R.sub.3 each independently are ##STR00025## wherein n>1, preferably wherein 1<n<10; or ##STR00026## wherein n>0, preferably wherein 0<n<10; or ##STR00027## wherein n>0, preferably wherein 0<n<10; or ##STR00028## wherein n>0, preferably wherein 0<n<10; and wherein R.sub.2 represents optionally substituted phenyl, optionally substituted benzyl, or optionally substituted phenethyl; and R.sub.4 represents straight or branched C.sub.1-6 alkyl, straight or branched C.sub.2-6 alkenyl, straight or branched C.sub.1-6 alkoxy, or straight or branched C.sub.1-6 alkanoyl.

18. The method according to claim 17, wherein the polyphosphonate copolymer comprises from 30-70 mol % A, and from 30-70 mol % B, preferably from 35-65 mol % A, and from 35-65 mol % B, most preferably from 40-60 mol % A, and from 40-60 mol % B, the total of A and B adding up to 100 mol % and wherein the polyphosphonate copolymer is a diblock copolymer, a normal tapered block copolymer, or a gradient copolymer, preferably wherein the polyphosphonate copolymer is a gradient copolymer which has a gradient as defined in Gleede et al., Macromolecules 2019, 52 (24), 9703-9714 of block(-like), medium or hard, preferably of medium or hard.

19. The method according to claim 18, wherein the polyphosphonate copolymer is a gradient copolymer which has a value relative to the length of the minority monomeric unit as defined by Shull, Interfacial activity of gradient copolymers, Macromolecules 2002, 35, 8631-8639 of 0.5-1, preferably 0.6-1, more preferably of 0.8-1 and wherein the polyphosphonate copolymer has a molecular weight (M.sub.n) between 1000 and 100.000, and a dispersity D between 1.01 and 10.00.

20. An aqueous suspension, comprising: micelles of the polyphosphonate copolymer of claim 14, wherein the micelles have a hydrodynamic radius R.sub.h as defined by ISO 22412:2008 of between 5 and 100 nm, a PDI of 0.001-0.5 as defined by ISO 22412:2008, and a concentration of between 1-500 mg/mL, preferably 5-490 mg/mL, more preferably 10-480 mg/mL.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] FIG. 1 depicts the chemical structure and a schematic representation of phenyl-co-ethyl phosphonate gradient co-polymer micelles.

[0069] FIG. 2 depicts the results of a toxicity study of phenyl-co-ethyl phosphonate gradient copolymer.

[0070] FIG. 3 depicts .sup.1H and .sup.31P MRI images of micelles of a block copolymer of PEG and PhPPn.

[0071] FIG. 4 depicts .sup.1H and .sup.31P MRI images of micelles of a block copolymer of PS and PEtPPn.

[0072] FIG. 5 depicts .sup.1H and .sup.31P MRI images of micelles of phenyl-co-ethyl phosphonate gradient copolymer.

[0073] FIG. 6 depicts the MRI results of injection of micelles according to the invention in the physalis.

DETAILED DESCRIPTION OF THE INVENTION

[0074] FIG. 1 depicts the chemical structure and a schematic representation of PhPPn-grad-EtPPn copolymer and its micelles.

[0075] FIG. 2 shows the result of a cell viability study with different concentrations of PhPPn.sub.30-grad-EtPPn.sub.30 micelles and monocytes, leukocytes and granulocytes.

[0076] FIG. 3 depicts MRI images of a PEG.sub.5000-b-P(PhPPn).sub.30 micelle, with from left to right: an image based on .sup.1H as a reference, and an image based on the frequency of .sup.31P. The concentration of micelles was 40 mg/mL, and the concentration of .sup.31P was 3.5 mg/mL. The SNR was 7.9.

[0077] FIG. 4 depicts MRI images of a PEtPPn.sub.62-b-PS.sub.345 micelle, with from left to right: an image based on 1H as a reference, and an image based on the frequency of .sup.31P. The concentration of micelles was 200 mg/mL, and the concentration of .sup.31P was 11 mg/mL. The SNR was 18.3.

[0078] FIG. 5 depicts MRI images of a PhPPn.sub.30-grad-EtPPn.sub.30 micelle, with from left to right: an image based on .sup.1H as a reference, an image based on the frequency of .sup.31P in EtPPn, an image based on the frequency of .sup.31P in PhPPn, and the .sup.31P sum image. The concentration of micelles was 38 mg/mL, and the concentration of .sup.31P was 7.7 mg/mL. The SNR was 26.4.

[0079] FIG. 6 depicts MRI images of PhPPn.sub.30-grad-EtPPn.sub.30 micelles injected into a physalis, with from left to right: an image based on 1H as a reference, the .sup.31P sum image, and a merged image showing the .sup.31P sum image overlayed on the .sup.1H based image. The arrow denotes the site of injection of the micelles.

EXAMPLES

Materials

[0080] Solvents and chemicals were purchased from Acros Organics, Sigma Aldrich or Fluka and used as received unless otherwise stated. All chemicals were purchased in the highest purities, dry and stored over molecular sieves (4 ), if possible. Ultrapure water with a resistivity of 18 M cm.sup.1 (Milli-Q, Millipore) was used for the self-assembly experiments. 2-(Benzyloxy)ethanol was acquired from ABCR, distilled from calcium hydride and stored over molecular sieves (3 and 4 ) and under inert gas prior to use. DBU was purchased from Sigma Aldrich, distilled from calcium hydride and stored over molecular sieves (3 and 4 ) and under inert gas prior to use. Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany) or Merck and used as received.

Methods

[0081] Size exclusion chromatography (SEC) measurements were performed in DMF (containing 1 g.Math.L.sup.1 of LiBr) at 60 C. and a flow rate of 1 mL mirn.sup.1 with a PSS SECcurity as an integrated instrument, including three PSS GRAM column (100/1000/1000 g mol.sup.1) and a refractive index (RI) detector. Calibration was carried out using poly(ethylene glycol) or polystyrene standards supplied by Polymer Standards Service. The SEC data were plotted with OriginPro 9 software from OriginLab Corporation.

[0082] NMR spectroscopy was measured at Brucker Avance III 400 MHz spectrometer equipped with a PA BBO 400S1 BBF-H-D-05 Z SP probe at 298 K. As deuterated solvents CDCl.sub.3, CD.sub.2Cl.sub.2 or D.sub.2O were used. The proton spectra were calibrated against the solvent signal (CDCl.sub.3: H=7.26 ppm, CD.sub.2Cl.sub.2: H=5.32 ppm, D.sub.2O: H=4.79 ppm). Longitudinal relaxation times T.sub.1 were measured using an inverse recovery sequence. For the measurements of transverse relaxation times T.sub.2 the Carr-Purcell-Meiboom-Gill (CPMG) sequence, as included in the Bruker Topspin software, was used. At least 10 data points were acquired, which were then used for data fitting. An interscan delay was set to 5T.sub.1. In case of samples in water, deuterium oxide (10 vol %) was added to the samples for locking. Data analysis was performed using Mestrenova14 from Mestrelab.

[0083] Dynamic Light Scattering (DLS) to measure R.sub.h was done at a Zetasizer Lab from Malvern UK at a scattering angle of 900, and 295 K. The samples were diluted with ultrapure water so that the attenuator was at the step 10-11 (set automatically by the device). Data analysis was done with ZSxplorer 2.2.0.147 software from Malvern Panalytical

[0084] Magnetic Resonance Imaging (M R) images were recorded at a vertical Bruker AVANCE.sup.III and AVANCE NEO 9.4T wide bore NMR spectrometers driven by ParaVision 5.1 and 360 v3.0, respectively, and operating at frequencies of 400.2 MHz for .sup.1H and 162.0 MHz for .sup.31P measurements. Experiments were carried out using a Bruker microimaging unit (Micro 2.5) equipped with actively shielded gradient sets (capable of 1.5 T/m maximum gradient strength and 150 s rise time at 100% gradient switching) and a dual tunable .sup.1H/.sup.31P 25-mm birdcage resonator. Due to the vertical orientation of the MRI system, all phantoms were scanned in an upright position.

[0085] Anatomical reference images were acquired by standard FLASH or RARE (FLASH: FOV 2020 mm.sup.2, matrix 128128, TE 1.62 ms, TR 60 ms, ST 2 mm, NA 1, TAcq 8 sec; RARE: FOV 3030 mm.sup.2, matrix 256256, TE 4.39 ms, TR 4 sec, ST 0.75 mm, RARE factor 4, NA 1, TAcq 4 min 16 sec). Subsequently, polyphosphonates were detected by using 2D .sup.31P chemical shift imaging (CSI) or multi-chemical selective imaging (mCSSI). The slice used for spectroscopic imaging (6-10 mm) was placed in axial orientation covering a large part of the phantom, or physalis. The 2D .sup.31P CSI data sets were recorded with a sine-bell acquisition-weighted sequence to improve the spatial response function using the following parameters: flip angle, 45; TR, 250 ms; matrix 3232; data points in the spectral domain, 1024; spectral width, 6510 Hz: slice selection with a 500-s sinc3 pulse; TAcq, 8 min. Data sets were analyzed by an in-house-developed software module.sup.17 based on the LabVIEW package (National Instruments, Austin). An exponential filter of 20 Hz was applied in the spectroscopic direction.

[0086] mCSSI was carried out as described previously.sup.18 using selective exciting frequencies with a bandwidth of 913 Hz (gauss 3 ms): For P(PhPPn.sub.30-grad-EtPPn.sub.30) 3030 163.0138883 and 162.0110640 MHz, PEG.sub.5000-b-P(PhPPn.sub.30) 162.0107971 MHz, PEtPPn.sub.62-b-PS.sub.345 162.0140139 MHz (TE 6.32 ms, TR 2.5 sec, RARE factor 32, matrix 3232, ST 8 mm, effective spectral bandwidth 15000 Hz, NA 200, TAcq 8 min 20 sec.

[0087] Ceil viabilityTo obtain circulating immune cells, heparinized blood was withdrawn by venous puncture of the inferior vena cava of a mouse. Blood was collected via a 23G cannula in heparin-aerated collection tubes. Erythrocytes were lysed by adding the 4-fold amount of ammonium chloride buffer (pH 7.4). After 10 min of incubation at room temperature the samples were centrifuged at 350g for 10 min at 20 C.

[0088] To determine cell toxicity of 3P micelles, 110.sup.6 murine immune cells were incubated with either 10 or 50 L/mL in DMEM and incubated for 1 h at 37 C. Afterwards, cells were washed and stained with CD45, CD11 b and Ly6G (all 1:100) to discriminate monocytes (CD45.sup.+, CD11b.sup.+, Ly6G.sup.), lymphocytes (CD45.sup.+, CD11 b.sup.+, Ly6G.sup.) and granulocytes (CD45.sup.+, CD11b.sup.+, Ly6G.sup.+). To determine the number of dead cells, samples were taken up in MACS buffer with 1 g/mL DAPL. Cells were gated with appropriated FSC/SSC settings and the number of DAPL.sup. cells were determined via flow cytometry.

[0089] Differential Scanning Calorimetry (DSC) measurements were performed using a Trios DSC 25 series thermal analysis system the temperature range from 80 C. to 500 C. under nitrogen with a heating rate of 10 C. min.sup.1. All glass transition temperatures (T.sub.g) were obtained from the second heating ramp of the experiment.

Monomer Synthesis

Ethyl phosphonic dichloride (EtPCI).sup.8

[0090] A mixture of diethyl-ethylphosphonate (252.9 g, 1.521 mol) and DMF (1.3 mL) were added dropwise to refluxing thionyl chloride (305 mL, 4.2 mol). Strong gas evolution of ethylene chloride and sulfur dioxide indicated the progress of the reaction. After 16 hours the gas evolution declined. To complete the reaction the bath temperature was increased to 120 C. for 24 h. The thionyl chloride was separated via distillation. Two times of fractionated distillation of the raw product yielded the desired dichloride as a yellowish liquid (202.2 g, yield 100%, bp 40-42 C./7.Math.10.sup.2 mbar).

[0091] .sup.1H NMR (CDCl.sub.3, ppm): =2.6 (dq, .sup.2J.sub.HP=15.0 Hz, .sup.3J.sub.HH=7.5 Hz, 2H, PCH.sub.2), 1.4 (dt, .sup.3J.sub.HP=30.1 Hz, .sup.3J.sub.HH=7.5 Hz, 3H,methyl group).

[0092] .sup.31P{H} NMR (CDCl.sub.3, ppm): =53.7.

2-Ethyl-2-oxo-1,3,2-dioxaphospholane (1) (EtPPn).SUP.8

[0093] A flame-dried three-necked round-bottom flask, equipped with a magnetic stirring bar and two dropping funnels, was charged with 400 mL of dry THF and cooled to 21 C. Ethylphosphonic dichloride (153.4 g, 1.04 mol) was dissolved in dry THF (400 mL) and transferred into one dropping funnel via a flame-dried stainless steel capillary. A solution of dry ethylene glycol (64.8 g, 1.04 mol) and dry pyridine (165.1 g, 2.08 mol) in THF (300 mL) was transferred into the second dropping funnel via a flame-dried stainless steel capillary. A slow dropping speed was adjusted to be approximately equal for both mixtures. After complete addition the solution was stirred for 1 h and kept overnight at 80 C. to facilitate the precipitation of the pyridinium hydrochloride byproduct. The precipitate was removed by filtration via a flame-dried Schlenk funnel, and the solvent was removed at reduced pressure. Two times of fractionated distillation yielded the desired product as colorless oil (86.3 g, yield 61%, bp 61 C./2.1.Math.10.sup.3 mbar).

[0094] .sup.1H NMR (CDCl.sub.3, ppm): =4.6-4.0 (m, 4H, CH.sub.2CH.sub.2), 1.9 (dq, .sup.2J.sub.HP=18.3 Hz, .sup.3J.sub.HH=7.8 Hz, 2H, PCH.sub.2), 1.1 (m, 3H, CH.sub.3).

[0095] .sup.31P{H} NMR (CDCl.sub.3, ppm): =52.5.

Methylphosphonic dichloride (MePCI).SUP.9

[0096] A mixture of dimethyl-methyl phosphonate (114.5 g, 0.92 mol) and DMF (0.9 mL) were added dropwise to refluxing thionyl chloride (160 mL, 2.2 mol). Strong gas evolution of methyl chloride and sulfur dioxide indicate the progress of the reaction. After 12 hours the gas evolution declined. To complete the reaction the bath temperature was increased to 120 C. for 24 h. The thionyl chloride was separated via distillation. Two times of fractionated distillation of the raw product yielded the desired dichloride as colorless crystals (34.7 g, yield: 28%, bp. 48-50 C./1.Math.10.sup.3 mbar).

[0097] .sup.1H NMR (, ppm): =2.5 (d, .sup.2J.sub.HP=16.4 Hz, 3H, CH.sub.3).

[0098] .sup.31P{H} NMR (CDCl.sub.3, ppm): =43.7

2-methyl-1,3,2-dioxaphospholane 2-oxide (4) (MePPn).SUP.9

[0099] A flame-dried three-necked round-bottom flask, equipped with a magnetic stirring bar and two dropping funnels, was charged with 100 mL of dry THF and cooled to 21 C. Methylphosphonic dichloride (34.7 g, 261 mmol) was dissolved in dry THF (250 mL) and transferred into one dropping funnel via a flame-dried stainless steel capillary. A solution of dry ethylene glycol (16.2 g, 261 mmol) and dry pyridine (41.3 g, 521 mmol) in THF (250 mL) was transferred into the second dropping funnel via a flame-dried stainless steel capillary. A slow dropping speed was adjusted to be approximately equal for both mixtures. After complete addition, the solution was stirred for 1 h and stored over night at 80 C. to facilitate the precipitation of the pyridinium hydrochloride byproduct. The precipitate was removed by filtration via a flame-dried Schlenk funnel and the solvent was removed at reduced pressure. Fractionated distillation yielded the desired product as colorless crystals (8.2 g, yield: 27%, b.p. 80C/1.Math.10.sup.2 mbar).

[0100] .sup.1H NMR (CDCl.sub.3, ppm): =4.5-4.1 (m, 4H, CH.sub.2CH.sub.2), 1.6 (d, .sup.3J.sub.HP=17.6 Hz, 3H, CH.sub.3).

[0101] .sup.31P {H} NMR (CDCl.sub.3, ppm): =48.7.

2-phenyl-1,3,2-dioxaphospholane 2-oxide (PhPPn)

[0102] PhPPn was performed according to a modified literature protocol.sup.9 A flame-dried three-necked round-bottom flask, equipped with a magnetic stirring bar and two dropping funnels, was charged with 100 mL of dry THF and cooled to 21 C. Phenylphosphonic dichloride (50.8 g, 260 mmol) was dissolved in dry THF (250 mL) and transferred into one dropping funnel via a flame-dried stainless steel capillary. A solution of dry ethylene glycol (16.2 g, 260 mmol) and dry pyridine (41.2 g, 521 mmol) in THF (250 mL) was transferred into the second dropping funnel via a flame-dried stainless steel capillary. A slow dropping speed was adjusted to be approximately equal for both mixtures. After complete addition, the solution was stirred for 1 h and stored over night at 80 C. to facilitate the precipitation of the pyridinium hydrochloride byproduct. The precipitate was removed by filtration via a flame-dried Schlenk funnel and the solvent was removed at reduced pressure. Fractionated distillation yielded the desired product as colorless solid (34.3 g, yield: 71%, b.p. 113-115 C./1.Math.10.sup.3 mbar).

[0103] .sup.1H NMR (CDCl.sub.3, ppm): =7.8 (dd, .sup.4J.sub.HP=14.2 Hz, .sup.3J.sub.HH=6.9 Hz, 2H, aromatic protons ortho), 7.6-7.4 (m, 3H, aromatic protons meta, para), 4.8-4.3 (m, 4H, OCH.sub.2CH.sub.2O)

[0104] .sup.31P{H} NMR (CDCl.sub.3, ppm): =36.0.

Example 1: PPn Gradient Copolymer Micelles

PPn Gradient Copolymer Synthesis

Ring-Opening Polymerization Catalyzed with DBU

[0105] Polymerization was performed according to a modified literature protocol..sup.10 The particular monomers were weighed in a flame-dried Schlenk-tube, dissolved in dry benzene and dried by lyophilization. The monomer was dissolved in dry dichloromethane to a total concentration of 4 mol L.sup.1. A stock solution of initiator 2-methoxyethanole in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1 and the calculated amount was added to the monomer solution. A stock solution of DBU in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1. The monomer solution and the catalyst solution were set to the respective reaction temperature (in general 10 C.).

[0106] The polymerization was initiated by the addition of the calculated volume of catalyst solution containing 3.0 equivalents of DBU in respect to the initiator. Polymerization was terminated by the rapid addition of an excess of formic acid dissolved in dichloromethane with a concentration of 20 mg mL.sup.1. The colorless, amorphous polymers were purified by two times precipitation in cold diethyl ether and dried in vacuo. Yields ranged from 70% to 95%.

[0107] Representative NMR data of P(PhPPn.sub.n-grad-MePPn.sub.m):

[0108] .sup.1H NMR (CDCl.sub.3, ppm): =7.9-7.6 (m, aromatic protons ortho), 7.6-7.3 (m, aromatic protons meta, para), 4.4-3.9 (m, backbone CH.sub.2), 3.3-3.2 (m, initiator CH.sub.3), 1.6-1.2 (m, PCH.sub.3)

[0109] .sup.13C {H} NMR(CDCl.sub.3, ppm): =132.9 (s, broad, aromatic-C-para), 131.7 (s, broad, aromatic-C-ortho), 128.6 (s, broad, aromatic-C-meta), 126.9 (d, .sup.1J.sub.CP=190.9 Hz), 65.4-63.7 (m, broad, backbone CH.sub.2), 11.2 (d, J.sub.CP=145.3 Hz,PCH.sub.3)

[0110] .sup.31P{H} NMR (CDCl.sub.3, ppm): =32.4 (PCH.sub.3), 19.9 (P-Ph)

[0111] Representative NMR data of P(PhPPn.sub.n-grad-EtPPn.sub.m):

[0112] .sup.1H NMR (CDCl.sub.3, ppm): =7.9-7.6 (m, aromatic protons ortho), 7.6-7.3 (m, aromatic protons meta, para), 4.4-3.9 (m, backbone CH.sub.2), 330-3.24 (s, broad, initiator CH.sub.3), 1.9-1.5 (in, PCH.sub.2), 1.3-0.9 (m, PCH.sub.2CH.sub.3)

[0113] .sup.13C {H} NMR(CDCl.sub.3, ppm): =132.8 (s, broad, aromatic-C-para), 131.8 (s, broad, aromatic-C-ortho), 128.9-128.2 (m, aromatic-C-meta), 126.9 (d, broad, .sup.1J.sub.CP 191.2 Hz), 65.2-63.8 (m, backbone CH.sub.2), 18.8 (d, .sup.1J.sub.CP=142.9 Hz, PCH.sub.2), 6.4 (s broad, PCH.sub.2CH.sub.3)

[0114] .sup.31P{H} NMR (CDCl.sub.3, ppm): =35.2 (P-Et), 19.8 (P-Ph)

Kinetic Measurements of Copolymerizations

[0115] To study the incorporation behavior of the different monomers during co-polymerization, a polymerization monomer mixture with the initiator prepared as mentioned above was transferred into a dry NMR tube under inert gas. This mixture was used to setup all NMR parameters (like shim and lock) at 263 K. The reaction was started by adding the calculated volume of catalyst solution (3 eq of DBU in respect to the initiator). The NMR tube was quickly placed in the NMR spectrometer and the experiments were started.

Determination of Reactivity Ratios of Copolymerizations

[0116] The reactivity ratios were calculated by different nonterminal models following the instructions of, Jaacks,.sup.11 Frey.sup.12 or BSL.sup.13 and the Meyer-Lowry.sup.14 model as a terminal model. The protocol of Gleede et. al. was followed, and all methods used data from 0 up to 70% conversion to determine reactivity values..sup.15 The average out of at least three models was used; the standard deviation was below 5%.

[0117] For P(PhPPn-EtPPn) r.sub.A is 23.90.6 and r.sub.B is 0.0400.002

[0118] For P(PhPPn-MePPn) rp % is 4.48 0.02and r.sub.B is 0.2220.001

T.SUB.g .Measurements by DSC

[0119] PhPPn.sub.30-grad-EtPPn.sub.30: 20 C. [0120] PhPPn.sub.30-grad-MePPn.sub.30: 10 C. [0121] PhPPn.sub.50-grad-MePPn.sub.50: 10 C.

Preparation of Micelles of Gradient Copolymers

[0122] Water-soluble gradient-co-polymers were dissolved in water at a desired concentration (usually 1-4 wt. %), mixed by vortex (Fisherbrand) and subsequently sonicated for 1 min in an ultrasonic bath (Branson) forming transparent, slightly opalescent dispersions.

[0123] Alternatively, polymers (1-4 wt. % of polymer related to final aqueous solution) were dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for one minute and stirred overnight in an open vial to remove acetone. This procedure can be used for encapsulation of hydrophobic cargo, such as hydrophobic drugs. In this case, the hydrophobic cargo is dissolved in acetone together with polymer in required dose.

[0124] Data on R.sub.h, PDI, T.sub.1 and T.sub.2 for the micelles of different gradient copolymers can be found in Table 1 and 2.

TABLE-US-00002 TABLE 1 Relaxation times of gradient-co-polymers. .sup.31P con- Phenyl- Ethyl- tent in R.sub.h T.sub.1, PH T.sub.2, PH T.sub.1, ET T.sub.2, ET units units polymer [nm] PDI [s] [s] [s] [s] 30 30 20 wt. % 9 0.04 0.9 0.09 1.8 0.9 44 56 20 wt. % 9 0.07 0.9 0.17 1.8 0.8 60 40 19 wt. % 11 0.02 1 0.06 1.8 0.8 30 70 21 wt. % 11 0.2 0.9 0.23 2.1 1.3 32 28 20 wt. % 8 0.04 0.9 0.13 1.7 0.8 29 31 20 wt. % 8 0.06 0.9 0.14 1.8 1.0

TABLE-US-00003 TABLE 2 Relaxation times of gradient-co-polymers. .sup.31P con- Phenyl- Methyl- tent in R.sub.h T.sub.1, PH T.sub.2, PH T.sub.1, Me T.sub.2, Me units units polymer [nm] PDI [s] [s] [s] [s] 30 30 22 wt. % 11 0.26 0.9 0.29 2.2 1.2 47 53 22 wt. % 9 0.22 0.9 0.27 2.2 1.2

[0125] The results of transverse relaxation T.sub.2 in Table 1 and 2 are calculated based on monoexponential analysis of the measurement data.

[0126] FIG. 2 shows the result of a cell viability study with PhPPn.sub.30-grad-EtPPn.sub.30 micelles and monocytes, leukocytes and granulocytes. Viability at different micelle concentrations did not substantially differ from the control samples. Thus, no negative influence on cell viability was observed.

[0127] FIG. 3 depicts .sup.1H and .sup.31P MRI of micelles of phenyl-co-ethyl phosphonate gradient co-polymer (30 phenyl units and 30 ethyl units, PhPPn.sub.30-grad-EtPPn.sub.30). Signals from both phenyl- and ethyl-phosphonate units can be distinguished based their chemical shift and added to a sum image forming the final MR image; better relaxation times from both monomers lead to a better signal.

[0128] Spatial localization of the imaging agents on an anatomical proton image is depicted in FIG. 4. For acquiring this image, a small amount of the micelles (around 4 mg) was injected in a physalis. As can be seen, the imaging agents can be localized on an anatomical proton image.

Example 2: PEG-b-PPn Block Copolymer Micelles

Synthesis of Block-Copolymer poly(ethylene glycol-b-PhPPn) PEG-b-P(PhPPn):

[0129] Polymerization was performed according to a modified literature protocol..sup.10 The PhPPn monomer were weighed in a flame-dried Schlenk-tube, dissolved in dry benzene and dried by lyophilization. The monomer was dissolved in dry dichloromethane to a total concentration of 4 mol L.sup.1. A stock solution of m-PEG.sub.110 in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1 and the calculated amount was added to the monomer solution. A stock solution of DBU in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1. The monomer solution and the catalyst solution were set to the respective reaction temperature (in general 0 C.).

[0130] The polymerization was initiated by the addition of the calculated volume of catalyst solution containing 3.0 equivalents of DBU in respect to the initiator. Polymerization was terminated by the rapid addition of an excess of formic acid dissolved in dichloromethane with a concentration of 20 mg mL.sup.1. The colorless, amorphous polymers were purified by two times precipitation in cold diethyl ether and dried in vacuo. Yields ranged from 80% to 96%.

Representative NMR data of PEG.sub.5000-b-P(PhPPn):

[0131] .sup.1H NMR (CDCl.sub.3, ppm): =7.8-7.6 (m, aromatic protons ortho), 7.6-7.4 (m, broad, aromatic protons para), 7.4-7.2 (m, broad, aromatic protons meta), 4.3-4.9 (m, backbone CH.sub.2), 3.6 (s, broad, PEG protons), 3.4 (s, initiator CH.sub.3)

[0132] .sup.31P{H} NMR (CDCl.sub.3, ppm): =19.8

Preparation of Micelles of Block Copolymers

[0133] Water-soluble gradient-co-polymers were dissolved in water at a desired concentration (usually 1-4 wt. %), mixed by vortex (Fisherbrand) and subsequently sonicated for 1 min in an ultrasonic bath (Branson) forming transparent, slightly opalescent dispersions.

[0134] Alternatively, polymers (1-4 wt. % of polymer related to the final aqueous dispersion) were dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for one minute and stirred overnight in an open vial to remove acetone.

[0135] Data on R.sub.h, PDI, T.sub.1 and T.sub.2 for the micelles of two different clock copolymers can be found in Table 3.

TABLE-US-00004 TABLE 2 Relaxation times of polyphenylphosphonate-b-polyethylene glycol PhPPn Repeat .sup.31P content in units polymer R.sub.h [nm] PDI T.sub.1 [s] T.sub.2 [s] 30 11 wt. % 10 0.05 1.2 0.05 50 9 wt. % 9 0.1 1.3 0.04

Example 3: PEtPPn-b-PS Colloids

PEtPPn-b-PS Synthesis.SUP.19

[0136] A representative procedure for the synthesis of a PEtPn macro-CTA is described:

Ethyl ethylene phosphonate (1 g, 7.35 mmol, 60 eq) and 2-cyano-5-hydroxypental-2-yl dodecyl carbonotrithioate (48 mg, 0.123 mmol, 1 eq) were dissolved in anhydrous dichloromethane (1.83 mL) in an oven-dried 4 mL vial equipped with a magnetic stirring bar. The reaction mixture was homogenized by stirring at 20 C. followed by the addition of DBU (55 L, 55.9 mg, 0.37 mmol, 3 eq) and the solution was stirred at room temperature for 1.5 h before the reaction mixture was quenched by the rapid addition of an excess of formic acid solution in dichloromethane (20 mg mL.sup.1). The crude product was purified by precipitation into cold diethyl ether (20 C.) three times, and drying in vacuo to yield PEtPnC.sub.62 macro-CTA as a yellow viscous liquid (0.95 g, 91%)
Representative NMR data of PEtPPn.sub.62 macro-CTA:

[0137] .sup.1H NMR (CDCl.sub.3, ppm): =4.31-4.18 (m, backbone CH.sub.2); 3.34 (t, J=7.4 Hz, initiator -CH.sub.2S), 1.88-1.78 (m, PCH.sub.2), 1.24-1.15 (m, PCH.sub.2CH.sub.3).

[0138] .sup.31P{H} NMR (CDCl.sub.3, ppm): =35.2

Synthesis of PEtPPn.SUB.62.-b-PS.SUB.345 .by Aqueous Emulsion Polymerization

[0139] PEtPn.sub.62 macro-CTA macroinitiator (129 mg, 0.01 mmol, 1 eq) and deionized water (2.73 g, 20 w/w %) were placed in a Schlenk flask and stirred until the macroinitiator was completely dissolved. A stock solution of VA-044 (10 mg mL-1) was prepared and VA-044 (1.57 mg, 0.002 mmol, 0.3 eq) was added to the reaction mixture. Styrene (0.53 g, 5.11 mmol, 350 eq) was weighed into a separate vial and added to the solution followed by stirring (1500 rpm) for 30 min. Then, the Schlenk flask was immersed in an ice bath and the solution was deoxygenized with nitrogen for 30 min and then immersed in an oil bath at 80 C. for 23h. Finally, the flask was placed in an ice bath and opened to air to terminate the polymerization.

[0140] .sup.1H NMR (CDCl.sub.3, ppm): =7.2-6.2 (m, aromatic protons); 4.4-4.1 (m, EtPPn backbone); 2.0-1.1 (m, PS backbone and EtPPn sidechain)

[0141] .sup.31P{H} NMR (CDCl.sub.3, ppm): =35.2

[0142] Data on R.sub.h, PDI, T.sub.1 and T.sub.2 for the micelles of the block copolymer can be found in Table 4.

TABLE-US-00005 TABLE 4 Relaxation times of PEtPPn-b-PS Et-PPn .sup.31P content in Repeat units polymer R.sub.h [nm] PDI T.sub.1 [s] T.sub.2 [s] 62 5 wt. % 47 0.1 2.3 0.49

Example 4: PhPPn-block-EtPPn

PPn Block Copolymer Synthesis

Ring-Opening Polymerization Catalyzed with DBU

[0143] Polymerization was performed according to a modified literature protocol..sup.10 The PhPPn and EtPPn monomer were weighed in two different flame-dried Schlenk-tubes, dissolved in dry benzene and dried by lyophilization. The monomers was dissolved in dry dichloromethane to a total concentration of 4 mol L.sup.1. A stock solution of initiator 2-methoxyethanole in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1 and the calculated amount was added to the PhPPn monomer solution. A stock solution of DBU in dry dichloromethane was prepared with a concentration of 0.2 mol L.sup.1. The monomer solution and the catalyst solution were set to the respective reaction temperature (in general 10 C.). The polymerization was initiated by the addition of the calculated volume of catalyst solution containing 3.0 equivalents of DBU in respect to the initiator.

[0144] After 2.5 h the EtPPn solution was added rapidly to the reaction tube. Polymerization was terminated by the rapid addition of an excess of formic acid dissolved in dichloromethane with a concentration of 20 mg mL.sup.1. The colorless, amorphous polymers were purified by two times precipitation in cold diethyl ether and dried in vacuo. Yields ranged from 70% to 90%.

Representative NMR Data of P(PhPPn.sub.n-b-EtPPn.sub.m):

[0145] .sup.1H NMR (CDCl.sub.3, ppm): =7.9-7.6 (m, aromatic protons ortho), 7.6-7.3 (m, aromatic protons meta, para), 4.4-3.9 (m, backbone CH.sub.2), 3.30-3.24 (s, broad, initiator CH.sub.3), 1.9-1.5 (m, PCH.sub.2), 1.3-0.9 (m, PCH.sub.2CH.sub.3)

[0146] .sup.13C {H} NMR(CDCl.sub.3, ppm): =132.8 (s, broad, aromatic-C-para), 131.8 (s, broad, aromatic-C-ortho), 128.9-128.2 (m, aromatic-C-meta), 126.9 (d, broad, .sup.1J.sub.CP=191.2 Hz), 65.2-63.8 (m, backbone CH.sub.2), 18.8 (d, .sup.1J.sub.CP=142.9 Hz, PCH.sub.2), 6.4 (s broad, PCH.sub.2CH.sub.3)

[0147] .sup.31P{H} NMR (CDCl.sub.3, ppm): =35.2 (P-Et), 19.8 (P-Ph)

Preparation of Micelles of Block Copolymers

[0148] Water-soluble gradient-co-polymers were dissolved in water at a desired concentration (usually 1-4 wt. %), mixed by vortex (Fisherbrand) and subsequently sonicated for 1 min in an ultrasonic bath (Branson) forming transparent, slightly opalescent dispersions.

[0149] Alternatively, polymers (1-4 wt. % of water used for dispersion) were dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for one minute and stirred overnight in an open vial to remove acetone.

[0150] Data on R.sub.h, PDI, T.sub.1 and T.sub.2 for the micelles of two different gradient copolymers can be found in Table 5.

TABLE-US-00006 TABLE 5 Relaxation times of block-co-polymers. .sup.31P con- Phenyl- Methyl- tent in R.sub.h T.sub.1, PH T.sub.2, PH T.sub.1, ET T.sub.2, ET units units polymer [nm] PDI [s] [s] [s] [s] 50 50 20 wt. % 11 0.05 1.1 0.05 1.9 1.0 20 80 21 wt. % 30-70 0.3 1.1 0.17 2.2 1.5 nm

LITERATURE

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