Phosphorus 31 MRI agent

Abstract

The present invention relates to 31 the use of a phosphorus-containing polymer for measuring P-MRI, wherein the phosphorus-containing polymer is selected from polyphosphate, polyphosphonate, poly(phosphine oxide), polyphosphazene, polyphosphinate, polyphosphoramidate, polyphosphorodiamidate, polyphosphoamide, polythionophosphate and polythionophosphonate. The present invention further relates to an aqueous suspension containing a polyphosphonate copolymer and micelles of the polyphosphonate copolymer.

Description

Technical Field

[0001] The present invention relates to 31 the use of phosphorus (P)-containing polymers for P-MRI measurements, and to aqueous suspensions comprising polyphosphonate copolymers and micelles of polyphosphonate copolymers.

Background Art

[0002] Magnetic resonance imaging (MRI) is an anatomical imaging technique that generates images based on the magnetic resonance properties of tissues. Unlike other techniques such as radioactive imaging (e.g., PET) or optical imaging, MRI does not involve ionizing radiation and is not limited by the penetration depth into tissues. The contrast of the images can be enhanced by using contrast agents, which are often gadolinium-based. Gd(III) is a paramagnetic ion that modulates the relaxation properties of the protons of water to display brighter spots with high Gd concentration (so-called T 1 weighted images). Such contrast agents have raised toxicological concerns due to the reported accumulation of gadolinium in the brain and tissues, and ecological concerns due to the increased gadolinium concentrations detected in rivers and oceans. Superparamagnetic iron oxide nanoparticles are alternative contrast agents, but these also show accumulation in organs, and therefore the FDA has withdrawn many iron oxide-based formulations.

[0003] Hot spot MRI has introduced the imaging of other nuclei such as fluorine-19 that function as selective "color" labels on anatomical proton images. Since the hot spot nuclei are directly detected, the signal from the hot spot nuclei can be quantified from the signal-to-noise ratio of the MRI image. Hot spot MRI is useful, for example, for visualizing the processes underlying cardiovascular diseases and inflammation, labeling immune cells, and detecting tumors. 1-7 Hot spot agents do not contain metals and can therefore be a solution to the problems of proton MRI contrast agents.

[0004] However, current hot spot agents are still far from ideal.19 MRI agents typically contain liquid perfluoroalkyl substances and polyfluoroalkyl substances (PFAS), which are typically formulated as nanoemulsions or nanoparticles. PFAS are highly chemically stable and do not break down in the body. Furthermore, they are hydrophobic and lipophobic, and their low solubility can lead to long-term accumulation in organs over a period of up to several months. Such compounds are also considered major environmental pollutants. Additionally, the presence of a PFAS phase can make it difficult to co-encapsulate additional cargo, such as drugs and other therapeutic molecules. In particular, in emulsions, additional cargo can only be encapsulated within a thin surfactant layer.

[0005] Phosphorus 31 is a non-radioactive NMR-active isotope of phosphorus with a nuclear spin of 1 / 2 and is naturally present at 100%. Phosphorus 31 is ubiquitous in biomolecules such as nucleic acids, for example. However, the sensitivity of phosphorus 31 is only about 8% of the sensitivity of hydrogen 1. Due to its low sensitivity and often very short relaxation times, most biomolecules become undetectable. Nevertheless, 31 P MR spectroscopy can be used to monitor phosphorylation in muscle and sense the pH of cells. As a result, 31 MRI scanners equipped with P coils have become readily available. However, to date, 31 the low sensitivity of the P nucleus has been a major challenge in the development of exogenous 31 P imaging agents. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] An object of the present invention is to overcome one or more of the above disadvantages or at least provide a useful alternative. A further object of the present invention is to provide an MRI hot spot agent that does not accumulate in the human body. A further object of the present invention is to provide an MRI hot spot agent that is biocompatible. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an MRI hot spot agent that is biodegradable. A further object of the present invention is to provide an31 To provide an MRI hot spot agent with an improved S / N ratio as compared to P. A further object of the present invention is to provide an MRI hot spot agent that can be used for encapsulating a drug and / or other therapeutic molecules.

Means for Solving the Problems

[0007] Therefore, the present invention provides the use of a P-containing polymer, preferably as a hot spot agent, 31 for measuring P MRI. The P-containing polymer is selected from polyphosphate, polyphosphonate, poly(phosphine oxide), polyphosphazene, polyphosphinate, polyphosphoramidate, polyphosphorodiamidate, polyphosphoamide, polythionophosphate and polythionophosphonate. Since all the 31 P atoms are localized within the polymer, a locally high P concentration is achieved. Furthermore, the polymers used according to the present invention all contain phosphorus-31 in their polymer backbones, and as a result, a particularly high phosphorus-31 concentration can be achieved. 31 P MRI images can be obtained via direct imaging such as mCSSI (multi-echo chemical shift selective imaging) or RARE (rapid acquisition with relaxation enhancement) or via spectroscopic imaging such as CSI (chemical shift imaging). Preferably, 31 P MRI images are obtained via direct imaging.

[0008] Preferably, the amount of P in the polymer is at least 3% by weight, more preferably at least 5% by weight, even more preferably at least 10% by weight, and most preferably at least 15% by weight.

[0009] Preferably, the P-containing polymer is included in a colloidal system (i.e., a state of subdivision meaning that the dispersed molecules or multimolecular particles in the medium have dimensions of approximately 1 nm to 1 μm in at least one direction, or discontinuities are found at distances of that order in the system; IUPAC, Compendium of Chemical Terminology, 2nd ed. (the “Gold Book”), Compiled by A.D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997), Online version (2019-) created by S.J. Chalk. ISBN 0-9678550-9-8, https: / / doi.org / 10.1351 / goldbook), such as micelles dispersed in a liquid medium. Preferably, the liquid medium is water or a physiological solution. Preferably, the P-containing polymer is included in micelles having a diameter of 1 nm to 1 μm, more preferably 2 nm to 500 nm. In the case of non-spherical micelles, the aforementioned diameter is the maximum diameter of the particles. This, in particular 31 ensures a high concentration of the P core and further overcomes the sensitivity problem. Colloidal systems are easy to inject into the human body and are often more biocompatible than systems with larger particles.

[0010] For the formation of such colloids, different P-containing polymers can be used. For example, the P-containing polymer can be insoluble in water. Such a hydrophobic P-containing polymer can be formed into an emulsion using an emulsifier. The P-containing polymer can also be water-soluble, in which case the P-containing polymer can be formed into a colloidal system by crosslinking of the polymer chains. The crosslinking can be chemical or due to physical interactions between the polymer chains. Amphiphilic copolymer assemblies contain at least two types of monomers. One or both of the monomers can contain P. The P-containing monomer can be relatively hydrophilic, and the other monomer can be relatively hydrophobic. When the relatively hydrophilic monomer contains P, the relatively hydrophobic monomer can be styrene. Alternatively, the P-containing monomer can be relatively hydrophobic and the other monomer can be relatively hydrophilic. In such a case, the hydrophilic monomer can be, for example, poly(ethylene glycol) (PEG). Alternatively, the P-containing polymer can be a copolymer of at least two P-containing monomers, in which case one of the monomers is relatively hydrophilic compared to the other P-containing monomer which is relatively hydrophobic.

[0011] In an aqueous solution, copolymers such as block copolymers and gradient copolymers can form core-shell micelles having a relatively hydrophobic core and a hydrophilic shell. Importantly, the spheres of the hydrophilic polymer can stabilize the hydrophobic core and other nanocarriers in a physiological environment, ensuring the so-called stealth effect.

[0012] Nanocarriers such as micelles containing hydrophobic polymers and / or amphiphilic copolymers can be further used to encapsulate additional cargoes such as hydrophobic drugs and other therapeutic agents that would otherwise be biologically unavailable.

[0013] Advantageously, the P-containing polymer is an amphiphilic polymer that forms core-shell micelles in an aqueous solution such as a physiological solution, and the hydrophilic shell contains a relatively rich amount of hydrophilic P-containing monomer units. The high mobility of the polymer chains in the hydrophilic shell and the hydrophobic core 31It has been demonstrated to be advantageous for the signal-to-noise ratio in P MRI imaging.

[0014] Preferably, the P-containing polymer is biocompatible and / or biodegradable, and biodegradable means that the P-containing polymer is completely decomposed into components that can be excreted from the human body within one year. More preferably, the P-containing polymer has both biocompatibility and biodegradability.

[0015] Preferably, the P-containing polymer has a T g below 37 °C, for example below 35 °C, preferably below 30 °C, more preferably below 25 °C or below 20 °C, g where T 1 is measured by heating the P-containing polymer at 10 °C / min. Such a polymer becomes in a "liquid-like" viscous state when injected into the body, i.e., at body temperature, and thus during MRI measurement. This has a favorable effect on the longitudinal and transverse relaxation times, T 2 and T g and determines the signal intensity in combination with the concentration. Most preferably, the P-containing polymer has a T

[0016] In MR spectroscopy, following a pulse that changes the orientation of the nuclear spin to a specific angle (typically 90°), the magnetization of the element precesses around the z-axis and at the same time returns to the equilibrium state along the z-axis. The transverse x and y components of M (the total magnetization of the spins) decay towards zero with the characteristic time T 2 . The longitudinal Z component returns to the starting position with the time constant T 1 .

[0017] The longitudinal relaxation time T 1 can be measured using an inversion recovery sequence (well-known to those skilled in the art and described, for example, in NMR in Biological Systems: From Molecules to Human, K.V.R. Chary, Girjesh Govil, Springer, 2008). The transverse relaxation time T 2For the measurement, the Carr-Purcell-Meiboom-Gill (CPMG) sequence (also described in the aforementioned publication), which is a standard sequence pre-installed in the NMR spectrometer, can be used.

[0018] The signal-to-noise ratio (SNR) in hot spot MRI depends on the T of the molecular properties 1 and T 2 and depends on. Typically, the shorter T 1 is and the longer T 2 is, the higher the contrast of the image becomes. Therefore, to obtain the optimal SNR, it is advantageous to make T 1 as short as possible and T 2 as long as possible.

[0019] Preferably, T 1 is 5 seconds or less, for example, 3 seconds or less or 2.5 seconds or less. Preferably, T 2 is at least 0.03 seconds, for example, at least 0.04 seconds or 0.045 seconds, etc.

[0020] Preferably, the P-containing polymer has a T of 0.05 seconds to 5 seconds 1 , a T of 0.03 seconds to 3 seconds 2 (where T 1 > T 2 ), more preferably a T of 0.1 seconds to 3 seconds 1 and a T of 0.04 seconds to 1.5 seconds 2 (where T 1 > T 2 ), more preferably a T of 0.5 seconds to 2.5 seconds 1 and a T of 0.045 to 1.5 2 (where T 1 > T 2 ), and T 1 and T 2 are the longitudinal and transverse relaxation times calculated using a single exponential decay fit.

[0021] Preferably, in the P-containing polymer 31The chemical shift of P is greater than 4 ppm, such as greater than 6 ppm or greater than 10 ppm, and is typically distinguishable from endogenous background molecules such as ATP and DNA that have lower chemical shifts. 31 The P chemical shift and the polymer can be reliably distinguished. Preferably, the chemical shift of the P-containing polymer is from 4 to 100 ppm, more preferably from 6 to 60 ppm, even more preferably from 10 to 50 ppm, and most preferably from 20 to 40 ppm.

[0022] Preferably, the P-containing polymer is a polyphosphonate (PPn). Polyphosphonates are biocompatible polymers belonging to the class of polyphosphoesters. The chemical shift of PPns can be adjusted from 10 to 200 ppm, and the 31 P chemical shift and the polymer can be reliably distinguished. For example, the 31 P chemical shift of ethyl-PPn is 37 ppm, and that of phenyl-PPn is 22 ppm.

[0023] Furthermore, polymers derived from the group of polyphosphoesters, such as the polyphosphonates presented in the present invention, are known to be excellent carriers for other cargos, such as certain hydrophobic cargos like pharmaceuticals, and other therapeutic molecules.

[0024] Furthermore, polyphosphoesters, and thus polyphosphonates, are biodegradable. Furthermore, the degradation rate can be adjusted as needed by adjusting the chemical composition.

[0025] Preferably, the polyphosphonate is a copolymer. More preferably, the polyphosphonate is an amphiphilic copolymer. Amphiphilic copolymers contain hydrophilic monomer units and lipophilic / hydrophobic monomer units. In an aqueous solution, such polymers can form micelles having a hydrophobic core and a hydrophilic shell.

[0026] Preferably, the polyphosphonate copolymer contains at least two monomer units A and B, Monomer unit A = [Chemical formula] and Monomer unit B = [Chemical formula] wherein R 1 and R 3 are each independently a) [Chemical formula] (where n > 1, preferably 1 < n < 10), or b) [Chemical formula] (where n > 0, preferably 0 < n < 10), or c) [Chemical formula] (where n > 0, preferably 0 < n < 10), or d) [Chemical formula] (where n > 0, preferably 0 < n < 10) and R 2 represents an optionally substituted phenyl group, an optionally substituted benzyl group or an optionally substituted phenethyl group, and R 4 represents a linear or branched C 1~6 alkyl group, a linear or branched C 2~6 alkenyl group, a linear or branched C 1~6 alkoxy or a linear or branched C 1~6 alkanoyl group.

[0027] More preferably, R 1 and R3 is a) where n = 2, R 2 represents a methylphenyl group, a dimethylphenyl group, an ethylphenyl group, a methylbenzyl group or a phenethyl group, and R 4 is a straight-chain or branched C 1~4 alkyl group, a straight-chain or branched C 2~4 alkenyl group, a straight-chain or branched C 1~4 alkoxy group or a straight-chain or branched C 1~4 alkanoyl group.

[0028] Most preferably, R 2 represents a phenyl group, and R 4 represents a methyl group or an ethyl group, preferably an ethyl group.

[0029] Preferably, the polyphosphonate copolymer contains 30 to 70 mol% of A and 30 to 70 mol% of B, preferably 35 to 65 mol% of A and 35 to 65 mol% of B, and most preferably 40 to 60 mol% of A and 40 to 60 mol% of B, and the total of A and B is 100 mol%.

[0030] Preferably, the polyphosphonate copolymer contains 10 to 1000 monomer units, more preferably 30 to 500 monomer units, and even more preferably 50 to 400 monomer units.

[0031] Preferably, the polyphosphonate copolymer has a molecular weight (M n ) of 1000 to 100,000 g / mol, more preferably 3000 to 80,000 g / mol, and even more preferably 5000 to 60,000 g / mol. Preferably, the polyphosphonate copolymer has a polydispersity D(M w / M n ) of 1.01 to 10.00, more preferably 1.02 to 8, and even more preferably 1.03 to 6.

[0032] Preferably, the polyphosphonate copolymer is a diblock copolymer, a normal tapered block copolymer or a gradient copolymer. In the case of a copolymer of a hydrophilic monomer such as monomer unit B and a hydrophobic monomer such as monomer unit A, the copolymer of the above composition forms a core-shell micelle structure in an aqueous solution. In the core-shell micelle structure, the core is relatively rich in the most hydrophobic monomer (in this case monomer unit A), and the shell is relatively rich in the most hydrophilic monomer (in this case monomer unit B).

[0033] An advantage of the core-shell micelle is that a hydrophobic cargo such as a pharmaceutical and other therapeutic agents can be added to the core. Furthermore, the image resolution is improved, particularly due to the high mobility of the hydrophilic units within the shell.

[0034] It has been proven to be particularly advantageous when the copolymer is a gradient copolymer. In this case, the higher the proportion of hydrophilic monomer units in the hydrophobic core, the higher the mobility of the polymer chains in the core, and the relaxation times T 1 and T 2 are favorably affected, thereby improving the image resolution of the monomer units within the core. Since the monomer units within the shell have high mobility, the imaging signal is also improved.

[0035] Specifically, the present invention is a polyphosphonate copolymer containing two monomer units A and B, Monomer unit A =

Chemical formula

Chemical formula

Chemical formula

[0036] More specifically, the present invention is a polyphosphonate copolymer containing two monomer units A and B, Monomer unit A =

Chemical formula

Chemical formula

Chemical formula

[0037] Gleede et al. (Gleede, T.; Markwart, J. C.; Huber, N.; Rieger, E.; Wurm, F. R., in: Competitive Copolymerization: Access to Aziridine Copolymers with Adjustable Gradient Strengths. Macromolecules 2019, 52(24), 9703 - 9714) defines four groups of gradient structures via the r - parameter (reactivity parameter), a common method for characterizing copolymers. For a statistically polymerized copolymer, the four groups are defined as Soft gradient with 0 < Δr ≤ 1.5 Medium gradient with 1.5 < Δr ≤ 7.5 Steep gradient with 7.5 < Δr ≤ 25 Block (like) with 25 < Δr where Δr is the difference between the reactivity parameter r A of monomer A and the reactivity parameter r B of monomer B, and the reactivity parameter is calculated by taking the average of at least three values calculated according to the instructions of the Jaacks, 11 Frey 12 , BSL 13 and / or Meyer - Lowry 14 models, and the standard deviation must be less than 5%.

[0038] Preferably, the polyphosphonate copolymer is a gradient copolymer having a block (like), medium or steep, preferably medium or steep gradient, as defined by Gleede et al.

[0039] Preferably, the polyphosphonate copolymer is a gradient copolymer, where r A is from 3.5 to 25.0, r B is from 0.02 to 0.5, more preferably, r A is from 4.0 to 24.7, r B is from 0.03 to 0.4, and most preferably, r A is from 4.3 to 24.4, r Bis from 0.03 to 0.25.

[0040] Shull, Interfacial activity of gradient copolymers, Macromolecules 2002, 35, 8631 - 8639 describes the gradient parameter λ of symmetric copolymers. The gradient parameter λ represents the length of the composition gradient relative to the total length of the copolymer. In an AB copolymer, when λ = 0, the copolymer is a conventional block copolymer consisting of separate blocks of A units and B units. When λ = 1, the composition changes smoothly along the chain from pure A to pure B.

[0041] Preferably, the polyphosphonate copolymer is a gradient copolymer having a λ value of from 0.1 to 1, more preferably from 0.2 to 0.99, and most preferably from 0.3 to 0.98, with respect to the length of the minority monomer units defined by Shull et al.

[0042] The present invention also relates to an aqueous suspension containing micelles of the polyphosphonate copolymer, preferably an aqueous physiological saline solution. Preferably, the micelles have a hydrodynamic radius R of 5 - 100 nm, more preferably 6 - 90 nm, and even more preferably 7 - 80 nm, as measured by ISO 22412:2008 h having.

[0043] Preferably, the PDI of the micelles as defined by ISO 22412:2008 is from 0.001 to 0.5, more preferably from 0.01 to 0.5, and even more preferably from 0.01 to 0.2.

[0044] Preferably, the concentration of the micelles in the aqueous suspension is at least 1 mg / mL, more preferably at least 5 mg / mL, and most preferably at least 10 mg / mL. The higher the concentration, the higher the visibility in MRI. It is particularly advantageous when the micelle concentration in the aqueous suspension is from 1 to 500 mg / mL, preferably from 5 to 490 mg / mL, and more preferably from 10 to 480 mg / mL.

[0045] The present invention relates to an aqueous suspension containing micelles of a polyphosphonate copolymer, wherein the micelles have an R as defined by ISO 22412:2008 of 5 to 100 nm h , a PDI as defined by ISO 22412:2008 of 0.001 to 0.5, and a concentration of 1 to 500 mg / mL, preferably 5 to 490 mg / mL, more preferably 10 to 480 mg / mL, and particularly relates to the aqueous suspension.

Brief Description of the Drawings

[0046]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0047] Figure 1 shows the chemical structure and schematic diagram of the PhPPn-grad-EtPPn copolymer and its micelles.

[0048] Figure 2 shows PhPPn at different concentrations 30 -grad-EtPPn 30 and shows the results of a cell viability study using micelles and monocytes, leukocytes, and granulocytes.

[0049] Figure 3 shows PEG 5000 -b-P(PhPPn) 30Shows the MRI images of micelles. From left to right, as a reference, 1 image based on 31 H and 31 image based on the frequency of

[0050] Figure 4 shows the MRI images of PEtPPn 62 -b-PS 345 micelles. From left to right, as a reference, 1 image based on 31 H and 31 image based on the frequency of

[0051] Figure 5 shows the MRI images of PhPPn 30 -grad-EtPPn 30 micelles. From left to right, as a reference, 1 image based on 31 H, 31 image based on the frequency of 31 P in EtPPn, 31 image based on the frequency of

[0052] Figure 6 shows the MRI images of PhPPn 30 -grad-EtPPn 30 when micelles are injected into vacuoles. From left to right, as a reference, 1 image based on 31 H, 1 total image of 31 P, and a composite image obtained by overlapping the image based on

Example

[0053] Materials Solvents and chemicals were purchased from Acros Organics, Sigma Aldrich, or Fluka and used as received unless otherwise stated. All chemicals were purchased at the highest purity available and, when possible, dried and stored over molecular sieves (4 Å). For self-assembly experiments, ultrapure water with a resistivity of 18 MΩ / cm (Milli-Q, Millipore®) was used. 2-(Benzyloxy)ethanol was obtained from ABCR, distilled from calcium hydride, and stored over molecular sieves (3 Å and 4 Å) under an inert gas until use. DBU was purchased from Sigma Aldrich, distilled from calcium hydride, and stored over molecular sieves (3 Å and 4 Å) under an inert gas until use. Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany) or Merck and used as received.

[0054] Method Size exclusion chromatography (SEC) measurements were performed at 60 °C with a flow rate of 1 mL / min in DMF (containing 1 g / L LiBr) using a PSS SECurity integrated instrument containing three PSS GRAM columns (100 / 1000 / 1000 g / mol) and a refractive index (RI) detector. Calibration was performed using poly(ethylene glycol) or polystyrene standards provided by Polymer Standards Service. SEC data were plotted using OriginPro 9 software from OriginLab Corporation.

[0055] NMR spectroscopy was measured at 298 K using a Brucker Avance III 400 MHz spectrometer equipped with a PA BBO 400S1 BBF-H-D-05 Z SP probe. As deuterated solvents, CDCl 3 、CD 2 Cl 2 or D 2 O was used. Proton spectra were calibrated against the solvent signal (CDCl 3 : δH = 7.26 ppm, CD 2 Cl 2: δH = 5.32 ppm, D 2 O: δH = 4.79 ppm). The longitudinal relaxation time T 1 was measured using an inversion recovery sequence. For the measurement of the transverse relaxation time T 2 , the Carr-Purcell-Meiboom-Gill (CPMG) sequence included in the Bruker Topspin software was used. At least 10 data points were acquired and used for data fitting. The interscan delay was set to 5 × T 1 . For samples in water, deuterium oxide (10% by volume) was added for fixation. Data analysis was performed using Mestrelab's Mestrenova 14.

[0056] R h Dynamic light scattering (DLS) for measuring

[0057] R was carried out at a scattering angle of 90° and 295 K using a Zetasizer Lab from Malvern, UK. The sample was diluted with ultrapure water so that the attenuator was at step 10 - 11 (automatically set by the device). Data analysis was performed using Malvern Panalytical's ZSxplorer 2.2.0.147 software. 1 H measurements were performed at a frequency of 400.2 MHz, 31 P measurements were recorded using vertical Bruker AVANCE III and AVANCE NEO 9.4T wide-bore NMR spectrometers operating at a frequency of 162.0 MHz. The experiments were carried out using an active shield gradient set (maximum gradient strength 1.5 T / m, capable of a 100% gradient switching rise time of 150 μs) and a dual-adjustable 1 H / 31 P 25 mm birdcage resonator-equipped Bruker Microimaging Unit (Micro2.5). Since the MRI system was arranged vertically, all phantoms were scanned in an upright position.

[0058] Anatomical reference images were acquired with standard FLASH or RARE (FLASH: FOV 20×20 mm 2 , matrix 128×128, TE 1.62 ms, TR 60 ms, ST 2 mm, NA 1, TAcq 8 s, RARE: FOV 30×30 mm 2 , matrix 256×256, TE 4.39 ms, TR 4 s, ST 0.75 mm, RARE factor 4, NA 1, TAcq 4 min 16 s). Subsequently, 2D 31 P chemical shift imaging (CSI) or multi-chemical-selective imaging (mCSSI) was used to detect polyphosphonates. The slices (6 - 10 mm) used for spectroscopic imaging were axially positioned to cover most of the phantom or the cavity. 2D 31 PCSI datasets were recorded with a sine-bell acquisition weighting sequence to improve the spatial response function using the following parameters: flip angle 45°, TR 250 ms, matrix 32×32, number of data points in the spectral region 1024 points, spectral width 6510 Hz, slice selection with a 500-μs sinc3 pulse, TAcq 8 min. The datasets were analyzed by a software module developed in-house based on the LabVIEW package (National Instruments, Austin 17 . An exponential filter of 20 Hz was applied in the spectroscopic direction.

[0059] mCSSI was performed as previously described 18 , using a selective excitation frequency with a bandwidth of 913 Hz (Gaussian 3 ms): P(PhPPn 30 -grad-EtPPn 30 ), at 3030 163.0138883 and 162.0110640 MHz, PEG 5000 -b-P(PhPPn 30 ), 162.0107971 MHz, PEtPPn 62 -b-PS 345162.0140139 MHz (TE 6.32 ms, TR 2.5 seconds, RARE factor 32, matrix 32×32, ST 8 mm, effective spectral bandwidth 15000 Hz, NA 200, TAcq 8 minutes 20 seconds.

[0060] Cell viability - To obtain circulating immune cells, heparinized blood was collected by venipuncture of the inferior vena cava of mice. The blood was collected into a heparinized air-vented blood collection tube via a 23G cannula. Red blood cells were lysed by adding four volumes of ammonium chloride buffer (pH 7.4). After incubation at room temperature for 10 minutes, the samples were centrifuged at 350×g for 10 minutes at 20 °C.

[0061] 31 To determine the cytotoxicity of P micelles, 1×10 6 mouse immune cells were incubated in DMEM at 10 or 50 μL / mL and incubated at 37 °C for 1 hour. Then, the cells were washed and stained with CD45, CD11b, Ly6G (all 1:100) to identify monocytes (CD45 + , CD11b + , Ly6G - ), lymphocytes (CD45 + , CD11b - , Ly6G - ), and granulocytes (CD45 + , CD11b + , Ly6G + ). To measure the number of dead cells, the samples were taken up in MACS buffer containing 1 μg / mL DAPI. The cells were gated with appropriate FSC / SSC settings, and the number of DAPI - cells was measured by flow cytometry.

[0062] Differential scanning calorimetry (DSC) measurements were performed using a Trios DSC 25 series thermal analysis system at a heating rate of 10 °C / min in a nitrogen atmosphere over a temperature range of -80 °C to 50 °C. All glass transition temperatures (T g ) were obtained from the second heating ramp of the experiment.

[0063] Monomer synthesis Ethylphosphonic dichloride (EtPCl) 8 A mixture of diethyl ethylphosphonate (252.9 g, 1.521 mol) and DMF (1.3 mL) was added dropwise to thionyl chloride (305 mL, 4.2 mol) under reflux. The vigorous evolution of ethylene chloride and sulfur dioxide indicated the progress of the reaction. After 16 hours, the evolution of gas decreased. To complete the reaction, the bath temperature was raised to 120 °C and the reaction was carried out for 24 hours. Thionyl chloride was separated by distillation. When the raw material was fractionally distilled twice, the desired dichloride was obtained as a yellowish liquid (202.2 g, yield 100%, boiling point 40 - 42 °C / 7·10 -2 mbar). 1 H NMR (CDCl 3 , ppm): δ = 2.6 (dq, 2 J HP = 15.0 Hz, 3 J HH = 7.5 Hz, 2H, -P-CH 2 -), 1.4 (dt, 3 J HP = 30.1 Hz, 3 J HH = 7.5 Hz, 3H, methyl group). 31 P{H} NMR (CDCl 3 , ppm): δ = 53.7.

[0064] 2-Ethyl-2-oxo-1,3,2-dioxaphospholane (1) (EtPPn) 8 A flame-dried three-neck round-bottom flask equipped with a magnetic stir bar and two dropping funnels was charged with 400 mL of dry THF and cooled to -21 °C. Dichloride ethylphosphonate (153.4 g, 1.04 mol) was dissolved in dry THF (400 mL) and transferred to the dropping funnel via a flame-dried stainless-steel capillary. A THF (300 mL) solution of dry ethylene glycol (64.8 g, 1.04 mol) and dry pyridine (165.1 g, 2.08 mol) was transferred to the second dropping funnel via a flame-dried stainless-steel capillary. The dropping rates were adjusted slowly so that both mixtures were approximately equal. After complete addition, the solution was stirred for 1 hour and kept at -80 °C overnight to promote the precipitation of pyridinium hydrochloride as a byproduct. The precipitate was removed by filtration with a flame-dried Schlenk funnel, and the solvent was removed under reduced pressure. The desired product was obtained as a colorless oil (86.3 g, yield 61%, boiling point 61 °C / 2.1·10 -3 mbar). 1 H NMR (CDCl 3 , ppm): δ = 4.6 - 4.0 (m, 4H, -CH 2 -CH 2 -), 1.9 (dq, 2 J HP = 18.3 Hz, 3 J HH = 7.8 Hz, 2H, -P-CH 2 -), 1.1 (m, 3H, -CH 3 ). 31 P{H} NMR (CDCl 3 , ppm): δ = 52.5.

[0065] Methylphosphonic dichloride (MePCl) 9 A mixture of dimethyl methylphosphonate (114.5 g, 0.92 mol) and DMF (0.9 mL) was added dropwise to thionyl chloride (160 mL, 2.2 mol) under reflux. The vigorous evolution of methyl chloride and sulfur dioxide indicated the progress of the reaction. After 12 hours, the evolution of gas decreased. To complete the reaction, the bath temperature was raised to 120 °C and the reaction was carried out for 24 hours. Thionyl chloride was separated by distillation. By fractional distillation of the raw materials twice, the desired dichloride was obtained as colorless crystals (34.7 g, yield 28%, boiling point 48 - 50 °C / 1·10 -3 mbar). 1 1H NMR (, ppm): δ = 2.5 (d, 2 J HP = 16.4 Hz, 3H, -CH 3 ). 31 31P{H} NMR (CDCl 3 , ppm): δ = 43.7

[0066] 2-Methyl-1,3,2-dioxaphospholane-2-oxide (4) (MePPn) 9 A flame-dried three-necked round-bottom flask equipped with a magnetic stir 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 to a dropping funnel via a flame-dried stainless-steel capillary. A THF (250 mL) solution of dry ethylene glycol (16.2 g, 261 mmol) and dry pyridine (41.3 g, 521 mmol) was transferred to a second dropping funnel via a flame-dried stainless-steel capillary. The dropping rate was adjusted slowly so that both mixtures were approximately equal. After complete addition, the solution was stirred for 1 hour and kept at -80 °C overnight to promote the precipitation of the by-product pyridinium hydrochloride. The precipitate was removed by filtration through a flame-dried Schlenk funnel, and the solvent was removed under reduced pressure. By fractional distillation, the desired product was obtained as colorless crystals (8.2 g, yield: 27%, boiling point 80 °C / 1·10 -2 mbar). 1 1H NMR (CDCl 3, ppm): δ = 4.5 - 4.1 (m, 4H, -CH 2 -CH 2 -), 1.6 (d, 3 J HP = 17.6 Hz, 3H, -CH 3 ). 31 P{H} NMR (CDCl 3 , ppm): δ = 48.7.

[0067] 2-Phenyl-1,3,2-dioxaphospholane 2-oxide (PhPPn) PhPPn was carried out according to the modified literature protocol. 9 A flame-dried three-necked round-bottom flask equipped with a magnetic stir bar and two dropping funnels was charged with 100 mL of dry THF and cooled to -21 °C. Dichlorophenylphosphonate (50.8 g, 260 mmol) was dissolved in dry THF (250 mL) and transferred to the 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 to the second dropping funnel via a flame-dried stainless steel capillary. The dropping rate was adjusted slowly so that both mixtures were approximately equal. After complete addition, the solution was stirred for 1 hour and kept at -80 °C overnight to promote the precipitation of pyridinium hydrochloride as a byproduct. The precipitate was removed by filtration through a flame-dried Schlenk funnel, and the solvent was removed under reduced pressure. The desired product was obtained as a colorless solid by fractional distillation (34.3 g, yield: 71%, boiling point 113 - 115 °C / 1·10 -3 mbar). 1 H NMR (CDCl 3 , ppm): δ = 7.8 (dd, 4 J HP = 14.2 Hz, 3 J HH = 6.9 Hz, 2H, aromatic proton - ortho), 7.6 - 7.4 (m, 3H, aromatic proton - meta, para), 4.8 - 4.3 (m, 4H, -O-CH 2 -CH 2 -O-) 31P{H} NMR (CDCl 3 , ppm): δ = 36.0.

[0068] Example 1: PPn Gradient Copolymer Micelles Synthesis of PPn Gradient Copolymer Ring-Opening Polymerization with DBU Catalyst The polymerization was carried out according to a modified literature protocol. 10 Specific monomers were weighed into a flame-dried Schlenk tube, dissolved in dry benzene, and dried by freeze-drying. The monomers were dissolved in dry dichloromethane to a total concentration of 4 mol / L. A stock solution of the initiator 2-methoxyethanol in dry dichloromethane was prepared at a concentration of 0.2 mol / L, and a calculated amount was added to the monomer solution. A stock solution of DBU in dry dichloromethane was prepared at a concentration of 0.2 mol / L. The monomer solution and the catalyst solution were each set to their respective reaction temperatures (generally -10 °C).

[0069] The polymerization was initiated by adding a calculated amount of the catalyst solution containing 3.0 equivalents of DBU relative to the initiator. The polymerization was terminated by rapidly adding an excess amount of formic acid with a concentration of 20 mg / mL dissolved in dichloromethane. The colorless amorphous polymer was purified by precipitation twice in cold diethyl ether and dried under vacuum. The yield was in the range of 70% - 95%.

[0070] P(PhPPn n -grad-MePPn m ) Representative NMR Data: 1 H NMR (CDCl 3 , ppm): δ = 7.9 - 7.6 (m, aromatic proton - ortho), 7.6 - 7.3 (m, aromatic proton - meta, para), 4.4 - 3.9 (m, main chain - CH 2 -), 3.3 - 3.2 (m, initiator - CH 3 ), 1.6 - 1.2 (m, P - CH 3 ) 13 C{H} NMR (CDCl 3, ppm): δ = 132.9 (s, broad, aromatic - C - para), 131.7 (s, broad, aromatic - C - ortho), 128.6 (s, broad, aromatic - C - meta), 126.9 (d, 1 J CP = 190.9 Hz), 65.4 - 63.7 (m, broad, main - chain - CH 2 -), 11.2 (d, 1 J CP = 145.3 Hz, P - CH 3 ) 31 P{H} NMR (CDCl 3 , ppm): δ = 32.4 (P - CH 3 ), 19.9 (P - Ph)

[0071] Typical NMR data of P (PhPPn n -grad - EtPPn m ): 1 H NMR (CDCl 3 , ppm): δ = 7.9 - 7.6 (m, aromatic protons ortho), 7.6 - 7.3 (m, aromatic protons meta, para), 4.4 - 3.9 (m, main - chain - CH 2 -), 3.30 - 3.24 (s, broad, initiator - CH 3 ), 1.9 - 1.5 (m, P - CH 2 -), 1.3 - 0.9 (m, P - CH 2 -CH 3 ) 13 C{H} NMR (CDCl 3 , ppm): δ = 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, 1 J CP = 191.2 Hz), 65.2 - 63.8 (m, main - chain - CH 2 -), 18.8 (d, 1 J CP = 142.9 Hz, P - CH 2 -), 6.4 (s, broad, P - CH 2 -CH 3 ) 31 P{H} NMR (CDCl 3 , ppm): δ = 35.2 (P-Et), 19.8 (P-Ph)

[0072] Kinetic measurement of copolymerization To investigate the incorporation behavior of different monomers during copolymerization, the polymerization monomer mixture containing the initiator prepared as described above was transferred to a dry NMR tube under an inert gas. Using this mixture, all NMR parameters (such as shim and lock) were set at 263 K. The reaction was initiated by adding a calculated amount of the catalyst solution (3 equivalents of DBU with respect to the initiator). The NMR tube was quickly placed into the NMR spectrometer to start the experiment.

[0073] Determination of copolymerization reactivity ratios The reactivity ratios were calculated by different non-terminal models according to the instructions of the models as terminal models. Jaacks, 11 Frey 12 or BSL 13 and Meyer-Lowry 14 According to the instructions of the models as terminal models, the reactivity values were determined using data with a conversion rate of 0 - 70% in the protocol and all methods of Gleede et.al. 15 The average value of at least three models was used, and the standard deviation was less than 5%.

[0074] For P(PhPPn-EtPPn), r A is 23.9 ± 0.6, and r B is 0.040 ± 0.002. For P(PhPPn-MePPn), r A is 4.48 ± 0.02, and r B is 0.222 ± 0.001. T measurement by DSC g Measurement PhPPn 30 -grad-EtPPn 30 : -20 °C PhPPn 30 -grad-MePPn 30 : -10 °C PhPPn50 -grad-MePPn 50 :-10 °C

[0075] Preparation of gradient copolymer micelles The water-soluble gradient copolymer was dissolved in water at a desired concentration (usually 1-4 wt%), mixed by vortex (Fisherbrand), and subsequently sonicated in an ultrasonic bath (Branson) for 1 minute to form a transparent and slightly milky dispersion.

[0076] Alternatively, the polymer (1-4 wt% polymer with respect to the final aqueous solution) was dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for 1 minute and stirred overnight in an open vial to remove the acetone. This procedure can be used for the encapsulation of hydrophobic cargos such as hydrophobic drugs. In this case, the hydrophobic cargo is dissolved in acetone together with the required amount of polymer.

[0077] R of micelles of different gradient copolymers h , PDI, T 1 and T 2 The data regarding can be found in Tables 1 and 2.

[0078]

Table 1

[0079]

Table 2

[0080] The transverse relaxation T 2 of Tables 1 and 2 are calculated based on the single-exponential analysis of the measurement data.

[0081] Figure 2 shows PhPPn 30 -grad-EtPPn 30The results of the cell viability study using micelles and monocytes, leukocytes, and granulocytes are shown. The viability at different micelle concentrations was not substantially different from the control sample. Thus, no adverse effect on cell viability was observed.

[0082] Figure 3 shows the 30 H and 30 micelles of phenyl-co-ethylphosphonate gradient copolymer (30 phenyl units and 30 ethyl units, PhPPn 1 -grad-EtPPn 31 ). Signals from both phenylphosphonate units and ethylphosphonate units are distinguished based on their chemical shifts and can be added to the total image forming the final MR image, and better signals can be obtained if better relaxation times are obtained from both monomers.

[0083] The spatial localization of the contrast agent on the anatomical proton image is shown in Figure 4. To acquire this image, a small amount of micelles (about 4 mg) was injected into the vacuole. As can be seen, the imaging agent can be localized on the anatomical proton image.

[0084] Example 2: PEG-b-PPn Block Copolymer Micelles Synthesis of block copolymer poly(ethylene glycol-b-PhPPn), PEG-b-P(PhPPn): The polymerization was carried out according to a modified literature protocol. 10 The PhPPn monomer was weighed into a flame-dried Schlenk tube, dissolved in dry benzene, and dried by freeze-drying. The monomer was dissolved in dry dichloromethane to a total concentration of 4 mol / L. A stock solution of m-PEG 110 in dry dichloromethane was prepared at a concentration of 0.2 mol / L, and the calculated amount was added to the monomer solution. A stock solution of DBU in dry dichloromethane was prepared at a concentration of 0.2 mol / L. The monomer solution and the catalyst solution were set at their respective reaction temperatures (generally -0 °C).

[0085] The polymerization was initiated by adding a calculated amount of catalyst solution containing 3.0 equivalents of DBU relative to the initiator. The polymerization was terminated by rapidly adding an excess amount of formic acid with a concentration of 20 mg / mL dissolved in dichloromethane. The colorless amorphous polymer was purified by precipitation twice in cold diethyl ether and dried under vacuum. The yield ranged from 80% to 96%.

[0086] Typical NMR data of PEG 5000 -b-P(PhPPn): 1 1H NMR(CDCl 3 , ppm): δ = 7.8 - 7.6 (m, aromatic proton - ortho), 7.6 - 7.4 (m, broad, aromatic proton - para), 7.4 - 7.2 (m, broad, aromatic proton - meta), 4.3 - 4.9 (m, main chain - CH 2 -), 3.6 (s, broad, PEG proton), 3.4 (s, initiator - CH 3 ) 31 31P{H} NMR(CDCl 3 , ppm): δ = 19.8

[0087] Preparation of micelles of block copolymers The water-soluble gradient copolymer was dissolved in water at a desired concentration (usually 1 - 4 wt%), mixed by vortex (Fisherbrand), and subsequently sonicated in an ultrasonic bath (Branson) for 1 minute to form a transparent and slightly milky dispersion.

[0088] Alternatively, the polymer (1 - 4 wt% of the polymer relative to the final dispersion) was dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for 1 minute and stirred overnight in an open vial to remove acetone.

[0089] R of micelles of two different block copolymers h , PDI, T 1 and T 2 The data regarding can be found in Table 3.

[0090]

Table 3

[0091] Example 3: PEtPPn-b-PS Colloid Synthesis of PEtPPn-b-PS 19 A representative synthesis procedure of PEtPn macro CTA is described. Ethyl ethylenephosphonate (1 g, 7.35 mmol, 60 equivalents) and 2-cyano-5-hydroxypent-2-yl dodecylcarbonotrithioate (48 mg, 0.123 mmol, 1 equivalent) were dissolved in anhydrous dichloromethane (1.83 mL) in an oven-dried 4 mL vial equipped with a magnetic stir bar. The reaction mixture was stirred at 20 °C to homogenize, and then DBU (55 μL, 55.9 mg, 0.37 mmol, 3 equivalents) was added. After stirring the solution at room temperature for 1.5 h, an excess amount of formic acid solution (20 mgm / L) in dichloromethane was rapidly added to quench the reaction mixture. The crude product was purified by precipitation three times in cold diethyl ether (-20 °C) and dried under vacuum to obtain PEtPn 62 Macro CTA (0.95 g, 91%) was obtained as a yellow viscous liquid.

[0092] PEtPPn 62 Representative NMR data of macro CTA: 1 1H NMR (CDCl 3 , ppm): δ = 4.31 - 4.18 (m, main chain -CH 2 -); 3.34 (t, J = 7.4 Hz, initiator α-CH 2 -S-), 1.88 - 1.78 (m, P-CH 2 -), 1.24 - 1.15 (m, P-CH 2 -CH 3 ). 31 31P{H} NMR (CDCl 3 , ppm): δ = 35.2

[0093] Synthesis of PEtPPn 62 -b-PS 345 by aqueous emulsion polymerization PEtPn 62 The macro-CTA macroinitiator (129 mg, 0.01 mmol, 1 equiv) 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 equiv) was added to the reaction mixture. Styrene (0.53 g, 5.11 mmol, 350 equiv) was weighed into a separate vial, added to the solution, and stirred (1500 rpm) for 30 min. Next, the Schlenk flask was immersed in an ice bath, the solution was deoxygenated with nitrogen for 30 min, and then immersed in an 80 °C oil bath for 23 h. Finally, the flask was placed in an ice bath and opened to the air to terminate the polymerization. 1 H NMR (CDCl 3 , ppm): δ = 7.2 - 6.2 (m, aromatic protons); 4.4 - 4.1 (m, EtPPn backbone); 2.0 - 1.1 (m, PS backbone and EtPPn side chains) 31 P{H} NMR (CDCl 3 , ppm): δ = 35.2

[0094] R of the micelles of the block copolymer h , PDI, T 1 and T 2 Data regarding can be found in Table 4.

[0095]

Table 4

[0096] Example 4: PhPPn-block-EtPPn PPn block copolymer synthesis Ring-opening polymerization with DBU catalyst The polymerization was carried out according to a modified literature protocol. 10The PhPPn monomer and the EtPPn monomer were weighed respectively into two different flame-dried Schlenk tubes, dissolved in dry benzene and freeze-dried. The monomer was dissolved in dry dichloromethane to a total concentration of 4 mol / L. A stock solution of a dry dichloromethane solution of 2-methoxyethanol as the initiator was prepared at a concentration of 0.2 mol / L, and the calculated amount was added to the PhPPn monomer solution. A stock solution of a dry dichloromethane solution of DBU was prepared at a concentration of 0.2 mol / L. The monomer solution and the catalyst solution were set at their respective reaction temperatures (generally -10 °C). Polymerization was initiated by adding a calculated amount of the catalyst solution containing 3.0 equivalents of DBU relative to the initiator.

[0097] After 2.5 hours, the EtPPn solution was rapidly added to the reaction tube. Polymerization was terminated by rapidly adding an excess amount of formic acid with a concentration of 20 mg / mL dissolved in dichloromethane. The colorless amorphous polymer was purified by precipitation twice in cold diethyl ether and vacuum dried. The yield was in the range of 70% - 90%.

[0098] P(PhPPn n -b-EtPPn m ) representative NMR data: 1 H NMR(CDCl 3 , ppm): δ = 7.9 - 7.6 (m, aromatic proton - ortho), 7.6 - 7.3 (m, aromatic proton - meta, para), 4.4 - 3.9 (m, main chain - CH 2 -), 3.30 - 3.24 (s, broad, initiator - CH 3 ), 1.9 - 1.5 (m, P - CH 2 -), 1.3 - 0.9 (m, P - CH 2 -CH 3 ) 13 C{H}NMR(CDCl 3 , ppm): δ = 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, 1 J CP = 191.2 Hz), 65.2 - 63.8 (m, main chain - CH2 -), 18.8 (d, 1 J CP = 142.9 Hz, P-CH 2 -), 6.4 (s broad, P-CH 2 -CH 3 ) 31 P{H} NMR (CDCl 3 , ppm): δ = 35.2 (P-Et), 19.8 (P-Ph)

[0099] Preparation of Micelles of Block Copolymers The water-soluble gradient copolymer was dissolved in water at a desired concentration (usually 1 - 4 wt%), mixed by vortex (Fisherbrand), and subsequently sonicated in an ultrasonic bath (Branson) for 1 minute to form a transparent and slightly milky dispersion.

[0100] Alternatively, the polymer (1 - 4 wt% of the water used for dispersion) was dissolved in 300 mg of acetone (VWR GPR recapture, 99%) and added to ultrapure water. The solution was sonicated for 1 minute and stirred overnight in an open vial to remove acetone.

[0101] R of micelles of two different gradient copolymers h , PDI, T 1 and T 2 The data regarding can be found in Table 5.

[0102]

Table 5

[0103] References 1. Senders, M. L.; Meerwaldt, A. E.; van Leent, M. M. T.; Sanchez - Gaytan, B. L.; van de Voort, J. C.; Toner, Y. C.; Maier, A.; Klein, E. D.; Sullivan, N. A. T.; Sofias, A. M.; Groenen, H.; Faries, C.; Oosterwijk, R. S.; van Leeuwen, E. M.; Fay, F.; Chepurko, E.; Reiner, T.; Duivenvoorden, R.; Zangi, L.; Dijkhuizen, R. M.; Hak, S.; Swirski, F. K.; Nahrendorf, M.; Perez - Medina, C.; Teunissen, A. J. P.; Fayad, Z. A.; Calcagno, C.; Strijkers, G. J.; Mulder, W. J. M., Probing myeloid cell dynamics in ischaemic heart disease by nanotracer hot - spot imaging. Nat. Nanotechnol. 2020, 15(5), 398 - 405. 2. Floegel, U.; Temme, S.; Jacoby, C.; Oerther, T.; Keul, P.; Flocke, V.; Wang, X.; Boenner, F.; Nienhaus, F.; Peter, K.; Schrader, J.; Grandoch, M.; Kelm, M.; Levkau, B., Multi - targeted 1H / 19F MRI unmasks specific danger patterns for emerging cardiovascular disorders. Nat. Commun. 2021, 12(1), 5847. 3. Higuchi, M.; Iwata, N.; Matsuba, Y.; Sato, K.; Sasamoto, K.; Saido, T. C., 19F and 1H MRI detection of amyloid β plaques in vivo. Nat. Neurosci. 2005, 8(4), 527 - 533. 4. Srinivas, M.; Morel, P. A.; Ernst, L. A.; Laidlaw, D. H.; Ahrens, E. T., Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model. Magn. Reson. Med. 2007, 58(4), 725 - 734. 5. Koshkina, O.; Lajoinie, G.; Bombelli, F. B.; Swider, E.; Cruz, L. J.; White, P.; Schweins, R.; Dolen, Y.; Dinther, E. v.; Riessen, N. K. v.; Rogers, S. E.; Fokkink, R.; Voets, I. K.; Eck, E. R. H. v.; Heerschap, A.; Versluis, M.; Korte, C. d.; Figdor, C.; Vries, I. J. M. d.; Srinivas, M., Multicore liquid perfluorocarbon-loaded multimodal nanoparticles for stable ultrasound and 19F MRI applied to in vivo cell tracking. Adv. Funct. Mater. 2019, 29(19), 1806485. 6. Zhang, C.; Moonshi, S. S.; Wang, W.; Ta, H. T.; Han, Y.; Han, F. Y.; Peng, H.; Kral, P.; Rolfe, B. E.; Gooding, J. J.; Gaus, K.; Whittaker, A. K., High F-Content Perfluoropolyether-Based Nanoparticles for Targeted Detection of Breast Cancer by 19F Magnetic Resonance and Optical Imaging. ACS Nano 2018, 12(9), 9162 - 9176. 7. Ahrens, E.T.; Bulte, J.W.M., Tracking immune cells in vivo using magnetic resonance imaging. Nat. Rev. Immunol. 2013, 13(10), 755 - 763. 8. Wolf, T.; Steinbach, T.; Wurm, F.R., A Library of Well-Defined and Water-Soluble Poly(alkyl phosphonate)s with Adjustable Hydrolysis. Macromolecules 2015, 48(12), 3853 - 3863. 9. Steinbach, T.; Ritz, S.; Wurm, F.R., Water-Soluble Poly(phosphonate)s via Living Ring-Opening Polymerization. ACS Macro Letters 2014, 3(3), 244 - 248. 10. Steinbach, T.; Ritz, S.; Wurm, F.R., Water-Soluble Poly(phosphonate)s via Living Ring-Opening Polymerization. Acs Macro Lett. 2014, 3(3), 244 - 248. 11. Jaacks, V., A novel method of determination of reactivity ratios in binary and ternary copolymerizations. Die Makromolekulare Chemie: Macromolecular Chemistry and Physics 1972, 161(1), 161 - 172. 12. Blankenburg, J.; Kersten, E.; Maciol, K.; Wagner, M.; Zarbakhsh, S.; Frey, H., The poly(propylene oxide-co-ethylene oxide) gradient is controlled by the polymerization method: determination of reactivity ratios by direct comparison of different copolymerization models. Polymer Chemistry 2019, 10(22), 2863 - 2871. 13. Beckingham, B.S.; Sanoja, G.E.; Lynd, N.A., Simple and accurate determination of reactivity ratios using a nonterminal model of chain copolymerization. Macromolecules 2015, 48(19), 6922 - 6930. 14. Meyer, V.E.; Lowry, G.G., Integral and differential binary copolymerization equations. Journal of Polymer Science Part A: General Papers 1965, 3(8), 2843 - 2851. 15. Gleede, T.; Markwart, J.C.; Huber, N.; Rieger, E.; Wurm, F.R., Competitive Copolymerization: Access to Aziridine Copolymers with Adjustable Gradient Strengths. Macromolecules 2019, 52(24), 9703 - 9714. 16. Ng, C.; Cousins, I. T.; DeWitt, J. C.; Gluege, J.; Goldenman, G.; Herzke, D.; Lohmann, R.; Miller, M.; Patton, S.; Scheringer, M.; Trier, X.; Wang, Z., Addressing Urgent Questions for PFAS in the 21st Century. Environ. Sci. Technol. 2021, 55(19), 12755 - 12765. 17. Floegel U, Jacoby C, Goedecke A, Schrader J. In vivo 2D mapping of impaired murine cardiac energetics in NO-induced heart failure. Magn Reson Med. 2007;57:50 - 8. 18. Floegel U, Temme S, Jacoby C, Oerther T, Keul P, Flocke V, Wang X, Boenner F, Nienhaus F, Peter K, Schrader J, Grandoch M, Kelm M, Levkau B. Multi-targeted 1H / 19F MRI unmasks specific danger patterns for emerging cardiovascular disorders. Nat Commun. 2021;12:5847. 19. Resendiz-Lara, D. A.; Wurm, F. R. Polyphosphonate-Based Macromolecular RAFT-CTA Enables the Synthesis of Well-Defined Block Copolymers Using Vinyl monomers; ACS Macro Lett. 2021, 10, 10, 1273 - 1279.

Claims

1. ３１ The use of a P-containing polymer for measuring P-MRI, wherein the P-containing polymer is selected from polyphosphate, polyphosphonate, poly(phosphine oxide), polyphosphazene, polyphosphine, polyphosphoramide, polyphosphodiamide, polyphosphoamide, polythionophosphate and polythionophosphonate. The polymer contains phosphorus-31 in its backbone, and the polymer has a Tg of less than 37°C.

2. The use according to claim 1, wherein the amount of P in the polymer is at least 3% by weight.

3. The polymer undergoes a T interval of 0.1 to 3 seconds. １ and T 0.04 seconds to 1.5 seconds ２ It has such that T1 > T2, and T １ and T ２ The use according to claim 1, wherein is the longitudinal and transverse relaxation time calculated using a single exponential decay.

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

5. The use according to claim 4, wherein the polyphosphonate is a copolymer.

6. The polyphosphonate copolymer comprises at least two monomer units A and B, Monomer unit A = 【Chemistry 1】 and Monomer unit B = 【Chemistry 2】 And in the formula, R １ and R ３ Each of them operates independently. a) 【Transformation 3】 (wherein the formula n > 1), or b) 【Chemistry 4】 (wherein the formula n > 0), or c) 【Transformation 5】 (where n > 0 in the formula), or d) 【Transformation 6】 (In the formula, n > 0) And, R ２ represents optionally substituted phenyl, optionally substituted benzyl, or optionally substituted phenethyl, and R ４ is a straight-chain or branched C １～６ alkyl, straight-chain or branched C ２～６ alkenyl, straight-chain or branched C １～６ alkoxy or straight-chain or branched C １～６ alkanoyl, the use according to claim 5.

7. R １ and R ３ This is a) for n=2, R ２ This represents methylphenyl, dimethylphenyl, ethylphenyl, methylbenzyl or phenethyl, and R ４ The use according to claim 6, wherein is a linear or branched C1-4 alkyl, a linear or branched C2-4 alkenyl, a linear or branched C1-4 alkoxy, or a linear or branched C1-4 alkanoyl.

8. The use according to claim 6, wherein the polyphosphonate copolymer contains 30 to 70 mol% of A and 30 to 70 mol% of B, and the total of A and B is 100 mol%.

9. The use according to claim 6, wherein the polyphosphonate copolymer is a diblock copolymer, a conventional tapered block copolymer, or a gradient copolymer.

10. The use according to claim 9, wherein the polyphosphonate copolymer is a steep-gradient copolymer in which the difference △r between the reactivity parameter of monomer A and the reactivity parameter of monomer B is greater than 1.5 and less than or equal to 25.

11. The polyphosphonate copolymer has a molecular weight (M) of 1,000 to 100,000. ｎ ) and the use according to claim 6, having a dispersion degree D of 1.01 to 10.

00.

12. A polyphosphonate copolymer comprising two monomer units A and B, Monomer unit A = 【Transformation 7】 and Monomer unit B = 【Transformation 8】 And in the formula, R １ and R ３ teeth, 【Chemistry 9】 And n = 2 to 4, R ２ represents phenyl, benzyl, or phenethyl. R ４ This represents methyl, ethyl, or propyl. The copolymer comprises 40 to 60 mol% of A and 60 to 40 mol% of B. The polyphosphonate copolymer is a diblock copolymer, a conventional tapered block copolymer, or a gradient copolymer.

13. R1 and R3 are, 【Chemistry 10】 And n = 2, R2 represents phenyl, The polyphosphonate copolymer according to claim 12, wherein R4 represents ethyl.

14. It is a gradient copolymer, r Ａ 4.3 to 24.4, and r Ｂ The polyphosphonate copolymer according to claim 12, wherein the ratio is 0.03 to 0.

25.

15. An aqueous suspension comprising micelles of a polyphosphonate copolymer according to claim 12, wherein the micelles have a hydrodynamic radius R defined by ISO 22412:2008, ranging from 5 to 100 nm. ｈ Aqueous suspension having a PDI of 0.001 to 0.5 as defined by ISO 22412:2008 and a concentration of 1 to 500 mg / mL.

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