Amphiphilic block copolymer, polymer nanomicelle and use

Abstract

An amphiphilic block copolymer, comprising a hydrophobic block and a hydrophilic block, wherein the hydrophobic block is a polydopamine amino acid, and the hydrophilic block is selected from at least one of polysarcosine, polyethylene glycol, polyvinylpyrrolidone, polyacrylic acid, polyacrylamide, poly-N-ethylglycine, polyglutamic acid, polymethyloxazoline, polyethyloxazoline, monomethyl ether polyethylene glycol, polyethylene oxide or polyvinyl alcohol. A polymer nanomicelle, which is a chelate obtained by chelating a phenolic hydroxyl on a side chain of the amphiphilic block copolymer with trivalent bismuth ions. A hyaluronic acid-modified iron-based polymer nanomicelle probe, comprising an iron-based nanomicelle and hyaluronic acid modified on the outer layer thereof, wherein the iron-based nanomicelle is obtained by chelating the amphiphilic block copolymer with a ferric ion compound.

Description

An amphiphilic block copolymer, polymer nanomicelles and their applications

[0001] This application claims priority to Chinese patent applications CN 202411498377.4 and CN 202411523884.9. Technical Field

[0002] This application relates to the field of medical imaging contrast agent technology, specifically to an amphiphilic block copolymer, polymer nanomicelles based on the copolymer, and the application of the nanomicelles in enhanced CT imaging and magnetic resonance imaging. Background Technology

[0003] X-ray computed tomography (CT) imaging is one of the most widely used diagnostic imaging techniques. It boasts advantages such as high tissue discrimination, high contrast, and uniform radiation dose distribution, and is widely used in the diagnosis and differential diagnosis of various diseases, including malignant tumors, soft tissue diseases, and bone and joint disorders. During CT scans, CT contrast agents are frequently used to enhance the signal difference between tissues and lesions, thereby improving image quality and increasing lesion detection rates and disease diagnosis. Currently, small-molecule iodine CT contrast agents, primarily iohexol and iodized oil, are the most widely used in clinical practice. However, small-molecule iodine CT contrast agents have drawbacks, including small molecular weight, short blood circulation time, short imaging time, significant toxicity, lack of tissue specificity, and iodine allergy.

[0004] Using various nanomaterials as CT contrast agents has been considered an effective solution to improve imaging quality and reduce imaging time. Furthermore, many metallic compounds with high atomic numbers, such as gold and bismuth, have been developed as good CT contrast agents. Bismuth, in particular, possesses characteristics such as a high X-ray attenuation coefficient, low toxicity, and low cost, and has been applied in CT contrast agent research. Over the past few decades, various bismuth-based contrast agents for CT imaging have been reported, such as Bi, Bi₂S₃, Bi₂Se₃, Bi₂O₃, BiF₃, BiOI, and other Bi materials. However, existing bismuth contrast agents generally suffer from drawbacks such as complex synthesis processes, poor biocompatibility, and short imaging times, hindering their clinical application.

[0005] Magnetic resonance imaging (MRI) is one of the most commonly used imaging techniques in clinical practice. It offers advantages such as high soft tissue resolution and no radiation exposure, and is widely used in the diagnosis of various diseases, including malignant tumors, cardiovascular diseases, and neurological disorders. Dynamic contrast-enhanced magnetic resonance lymphography (DCE-MRL) is a technique for imaging and diagnosing the anatomical structure and pathology of lymphatic vessels. DCE-MRL can display the fine structure of lymphatic vessels and detect subtle abnormal lymphatic flow, aiding in the diagnosis of lymphatic-related diseases such as lymphorrhea and lymphangioma.

[0006] DCE-MRL generally requires the injection of magnetic resonance contrast agents to enhance lymphatic vessels and lymph nodes. Gadolinium-based contrast agents are most commonly used clinically, such as gadopentetate dimeglumine (Gd-DTPA, trade name Magnevisist) and gadoterate dimeglumine (Gd-DOTA, trade name Dotarem). However, the injection of contrast agents in DCE-MRL is a complex procedure. For lower extremity lymphatic imaging, the contrast agent is typically injected slowly between multiple toes, a time-consuming process requiring local anesthesia. For central lymphatic imaging, it requires injection through bilateral inguinal lymph nodes using a specialized needle under fluorescein endoscopy or ultrasound guidance, a method that demands highly skilled interventional personnel. The high in vivo clearance rate of gadolinium-based contrast agents also shortens the examination time window for DCE-MRL. Taking central lymphatic imaging as an example, the contrast agent enters the retroperitoneal lymphatic vessels approximately 2 minutes after injection, enters the cisterna chyli within 3-6 minutes, then the thoracic duct, and terminates at the venous angle within 5-9 minutes. Therefore, the examination time window for DCE-MRL is usually limited to within 15 minutes, making long-term imaging impossible. In addition, clinical studies have shown that gadolinium-based contrast agents pose a risk of gadolinium leakage, potentially inducing renal systemic fibrosis in patients with renal impairment, and are prone to long-term deposition in tissues and organs such as the brain, bones, and liver.

[0007] In summary, although CT imaging and magnetic resonance imaging (MRI) are two core technologies in the field of medical diagnostic imaging, existing contrast agents have many limitations:

[0008] In the field of CT imaging, the mainstream small-molecule iodine contrast agents (such as iohexol) have problems such as small molecular weight, short blood circulation time, narrow imaging window, significant toxic side effects, and the risk of iodine allergy. Bismuth has become a hot topic in CT contrast agent research and development due to its high X-ray attenuation coefficient, low toxicity, and low cost. However, existing bismuth-based contrast agents have complex synthesis processes, poor biocompatibility, and short imaging time, making clinical translation difficult.

[0009] In the field of magnetic resonance lymphatic imaging: Gadolinium-based contrast agents commonly used in clinical practice (such as Gd-DTPA) pose a risk of gadolinium leakage, which may induce renal systemic fibrosis in patients with renal impairment. Furthermore, their rapid clearance rate in the body limits the time window for dynamic contrast-enhanced magnetic resonance lymphatic imaging (DCE-MRL) examinations to typically within 15 minutes, failing to meet the demands for long-duration imaging. In addition, existing iron-based MRI contrast agents lack lymphatic targeting, making it impossible to achieve lymphatic system imaging through simple subcutaneous or intramuscular injection. The administration procedures are complex (requiring intra-lymph node injection or local anesthesia) and require highly skilled operators.

[0010] Chinese patent document CN110496231A discloses an amphiphilic polymer nanomicelle containing polydopamine amino acids chelating ferric ions and its application. This amphiphilic polymer nanomicelle chelates ferric ions using a catechol structure with biodegradable polydopamine amino acid block side chains. The preparation method includes: complexing an amphiphilic polymer containing polydopamine amino acids with a ferric ion compound, and obtaining the amphiphilic polymer nanomicelle containing polydopamine amino acids chelating ferric ions by solvent displacement. The micelles prepared by this invention can be used as a contrast agent for magnetic resonance T1 imaging, and their longitudinal relaxation rate (r1) is 5.6 mM. -1 s -1 The drug can circulate in mice for up to 150 minutes and produces significant results in magnetic resonance imaging (MRI). However, it lacks lymphatic targeting and cannot be used for imaging the lymphatic system. Furthermore, intramuscular and subcutaneous injections are more complex than intralymphatic injections. Even using substances that can bind to receptors in lymphatic vessels as contrast agents, intramuscular and subcutaneous injections may not necessarily result in lymph node imaging, further increasing the technical difficulty of lymphatic system-targeted imaging. Moreover, there is no universally available technology for both MRI and CT imaging.

[0011] Currently, there is an urgent need to develop a multifunctional polymer nanoprobe that can cover both CT and MRI imaging needs, and is simple to prepare, highly biosafe, has excellent imaging performance, and is convenient to administer, in order to solve the pain points of existing technologies. Summary of the Invention

[0012] The technical problem to be solved by this application is to provide an amphiphilic block copolymer, polymer nanomicelles and their applications, in response to the problems existing in the background art.

[0013] The technical solution adopted by this application to solve the above-mentioned technical problems is:

[0014] In a first aspect, this application provides an amphiphilic block copolymer comprising hydrophobic blocks and hydrophilic blocks; characterized in that the hydrophobic blocks are polydopa amino acids, the length of which is any integer between 1 and 200, more preferably 10-50.

[0015] The hydrophilic block is selected from at least one of polysarcosine, polyethylene glycol, polyvinylpyrrolidone, polyacrylic acid, polyacrylamide, poly-N-ethylglycine, polyglutamic acid, polymethyl oxazoline, polyethyl oxazoline, monomethyl ether polyethylene glycol, polyethylene oxide, or polyvinyl alcohol, and the number of its structural units is any integer between 1 and 2000, more preferably 50-200.

[0016] Furthermore, the topological structure of the amphiphilic block copolymer includes diblock, triblock, multiblock, random, star, ring, or grafted structures, preferably diblock.

[0017] Furthermore, the structure of the polymer is shown in formula (1) and / or formula (2):

[0018] In formulas (1) and (2), R1 is independently selected from alkyl, benzyl, or silyl groups;

[0019] m is the number of hydrophilic block structural units, which takes any integer value between 1 and 2000, and is more preferably 50-200;

[0020] n is the length of the polydopa amino acid chain segment, which takes any integer value between 1 and 200, and is more preferably 10-50.

[0021] In a second aspect, this application provides a polymer nanomicelle, wherein the polymer nanomicelle is a chelate in which the phenolic hydroxyl groups of the polymer side chain chelate trivalent bismuth ions, wherein the polymer is the amphiphilic block copolymer described in the first aspect;

[0022] The structure of the chelate is as follows:

[0023] The molar ratio of phenolic hydroxyl groups to trivalent bismuth ions in the polymer nanomicelles is 3:1 to 2;

[0024] Furthermore, the trivalent bismuth ion compound is selected from at least one of bismuth nitrate, bismuth chloride, bismuth sulfate, and bismuth ammonium citrate.

[0025] Furthermore, the particle size of the polymer nanomicelles is 10-500 nm, preferably 10-100 nm.

[0026] Furthermore, in the polymer nanomicelles, the bismuth ion loading efficiency is ≥50%, and the bismuth ion content is ≥15wt%.

[0027] Furthermore, the connection length of the hydrophilic block is preferably 50 to 200, and the connection length of the hydrophobic block is preferably 1 to 50.

[0028] Furthermore, the trivalent bismuth ion can be replaced by at least one of tantalum ion, tungsten ion, gadolinium ion, holmium ion, ytterbium ion, gold ion, lutetium ion, yttrium ion, rhenium ion, and actinium ion.

[0029] Thirdly, this application provides the use of the polymer nanomicelles described in the second aspect in the preparation of computed tomography imaging agents.

[0030] Fourthly, this application provides a CT contrast agent comprising the polymer nanomicelles described in the second aspect, and medically or pharmaceutically acceptable excipients, including but not limited to excipients and diluents. In some embodiments, the CT contrast agent can be administered orally or parenterally (primarily intravenously) to humans and animals. The dosage varies depending on the recipient, CT imaging site, dosage form, route of administration, etc.

[0031] Fifthly, this application provides a method for preparing the polymer nanomicelles described in the second aspect. The preparation method involves complexing a polymer with a trivalent bismuth ion compound and obtaining polymer nanomicelles containing trivalent bismuth ions via a solvent displacement method.

[0032] Specifically, the following steps are included:

[0033] (1) Preparation of amphiphilic block copolymers according to any one of claims 1 to 3: polydopa block-hydrophilic block copolymers are obtained by ring-opening polymerization of N-substituted thioanhydrides with dopa amino acid monomers and hydrophilic monomers.

[0034] (2) Preparation of CT imaging nanomicelles by solvent displacement method: The copolymer obtained in step (1) is dissolved in DMSO, and the trivalent bismuth ion compound of claim 5 is added, wherein the molar ratio of phenolic hydroxyl group to trivalent bismuth ion is 3:1 to 2. After dialyzing with deionized water, the mixture is filtered and the volume is adjusted to obtain the polymer nanomicelles.

[0035] In a sixth aspect, this application provides a hyaluronic acid-modified iron-based polymer nanomicelle probe, comprising iron-based nanomicelles and hyaluronic acid modified on the outer layer thereof; the iron-based nanomicelles are obtained by chelating the amphiphilic block copolymer described in the first aspect with a trivalent iron ion compound; the modification of the outer layer of the iron-based nanomicelles with hyaluronic acid is achieved by solvent exchange; the mass ratio of hyaluronic acid to iron-based nanomicelles is 2-10:90-98, and the molecular weight of the hyaluronic acid is 1-1000 kDa, preferably between 5-100 kDa, and more preferably between 10-100 kDa.

[0036] Furthermore, the connection length of the hydrophilic block is 50-200, more preferably 100-180, and even more preferably 108-165; the connection length of the hydrophobic block is 1-50, more preferably 10-30, and even more preferably 12-15.

[0037] Furthermore, the iron-based nanomicelles are spherical, cylindrical, or vesicle-shaped assemblies; the amphiphilic block copolymer has a linear, star-shaped, grafted, or hyperbranched structure.

[0038] Furthermore, the iron-based nanomicelles are neutral to slightly negatively charged, and the iron-based nanomicelles are connected to hyaluronic acid through multiple hydrogen bond interactions between the polyhydroxyl groups of hyaluronic acid (hydrogen bond donors) and the polyamino acid amide bonds (hydrogen bond acceptors) of the iron-based nanomicelle shell. In this application, the surface of the iron-based nanomicelles is hydrophilic, coated with water-soluble hyaluronic acid, and there is no ligand exchange process between the two. Moreover, the iron-based nanomicelles in this application are neutral to slightly negatively charged, and the surface of hyaluronic acid is also negatively charged. Therefore, the iron-based nanomicelles and hyaluronic acid cannot be connected through electrostatic interactions, and must be prepared through the specific "multiple hydrogen bond interactions between the polyhydroxyl groups of hyaluronic acid (hydrogen bond donors) and the polyamino acid amide bonds (hydrogen bond acceptors) of the iron-based nanomicelle shell" as described in this application.

[0039] Furthermore, the hyaluronic acid-modified iron-based polydopa amino acid nanoprobes provided in this application, as a contrast agent for T1-enhanced magnetic resonance lymphatic imaging, need to have a particle size smaller than the interstitial lymphatic space and larger than the intervascular space, i.e., a particle size between 6-500 nm. Since particle size variations affect the magnetic resonance enhancement effect, preferably, the average particle size of the nanomicelles should be between 10-30 nm. The iron loading concentration of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobes provided in this application as a contrast agent for T1-enhanced magnetic resonance lymphatic imaging should be between 10 and 20,000 ppm.

[0040] Furthermore, the trivalent iron ion can be replaced by at least one of tantalum ion, tungsten ion, gadolinium ion, holmium ion, ytterbium ion, gold ion, lutetium ion, yttrium ion, rhenium ion, and actinium ion.

[0041] The T1-enhanced magnetic resonance lymphocyte imaging contrast agent provided in this application can be used in humans and non-human mammals (such as mice, rats, guinea pigs, rabbits, pigs, dogs, etc.). Administration methods include subcutaneous injection, intramuscular injection, and intralymph node injection. Subcutaneous injection is preferred due to its advantages of simple operation, small required dose, long imaging time, and minimal trauma to the human body. The dosage varies depending on the recipient and administration method, but the overall dose required for magnetic resonance lymphocyte imaging is extremely low.

[0042] In a seventh aspect, this application provides the application of the hyaluronic acid-modified iron-based polymer nanomicelle probe described in the sixth aspect in the imaging of targeted lymphatic vessels and targeted lymph nodes.

[0043] In a seventh aspect, this application provides a method for preparing a hyaluronic acid-modified iron-based polymer nanomicelle probe as described in the sixth aspect, comprising the following steps:

[0044] (1) Preparation of the amphiphilic block copolymer according to claim 1: polydopa block-hydrophilic block copolymer is obtained by ring-opening polymerization of N-substituted thioanhydride with dopa amino acid monomer and hydrophilic monomer.

[0045] (2) Preparation of nanoprobes for magnetic resonance lymphocyte imaging by solvent exchange method:

[0046] (2.1) The copolymer obtained in step (1) is added to DMSO and stirred until dissolved;

[0047] (2.2) Add ferric ion compound, stir and dialyze with deionized water for 24 h to obtain iron-based nano micelle solution;

[0048] (2.3) Adjust the pH of the iron-based nanomicelle solution to 4.0-5.0, slowly add hyaluronic acid solution, and stir continuously to finally obtain a nanoprobe for magnetic resonance lymphoma imaging.

[0049] The beneficial effects of this application are:

[0050] (1) In CT imaging, the polymer nanomicelles provided in this application are easy to prepare, have moderate particle size, good dispersibility, and stable structure. They possess excellent biocompatibility and biodegradability, and can be applied to X-ray computed tomography imaging. The CT enhancement effect is far superior to common small-molecule iodine contrast agents, with a CT contrast efficiency approximately three times that of clinically used iohexol, and a higher contrast ratio compared to other contrast agents in the prior art. Furthermore, according to literature reports, Mn 2+ Ag + Plasma has a weak binding to the catechol structure and cannot be used to prepare polymer nanomicelles. Compared with other metal ions, the trivalent bismuth ions in this application have a better chelation effect with the catechol structure of the polymer, and the encapsulation efficiency of trivalent bismuth ions in polymer nanomicelles is higher.

[0051] (2) In terms of magnetic resonance imaging, this application employs highly biosafety nanomicelles assembled from iron-based nanomicelles obtained by chelating a hydrophobic polymer with polydopamine amino acids as hydrophobic blocks and trivalent iron ions, and hyaluronic acid, thus avoiding the biosafety risks caused by gadolinium leakage in traditional gadolinium-based contrast agents. Furthermore, the hyaluronic acid-modified iron-based polydopamine amino acid nanoprobes of this application, as a T1-enhanced magnetic resonance lymphatic imaging contrast agent, possess lymphatic vessel and lymph node targeting effects, with a residence time in the lymphatic system exceeding 4 hours. This specific contrast agent enhances the T1 enhancement effect of magnetic resonance lymphatic imaging and extends the examination time window, resulting in a comprehensive imaging effect superior to commercially available gadolinium-based contrast agents. In addition, the hyaluronic acid-modified iron-based polydopamine amino acid nanoprobes used in this application, as a T1-enhanced magnetic resonance lymphatic imaging contrast agent, can be administered via subcutaneous or intramuscular injection, in addition to intra-lymph node injection. The administration method is simpler, faster, and safer than commercially available gadolinium-based contrast agents. Attached Figure Description

[0052] Figure 1 is a TEM image of the polymer nanomicelles prepared in Example 1.

[0053] Figure 2 shows the dynamic light scattering pattern of the polymer nanomicelles prepared in Example 1.

[0054] Figure 3 shows the in vitro CT imaging of the polymer nanomicelles prepared in Example 1 and its intensity-concentration relationship.

[0055] Figure 4 shows the in vitro CT imaging of iohexol and its intensity-concentration relationship.

[0056] Figure 5 shows the transverse, coronal, and three-dimensional reconstructed images of the polymer nanomicelles prepared in Example 1 by intravenous injection of 120 mg Bi / kg in rabbits (before injection and 30 min after injection).

[0057] Figure 6 shows the transverse, coronal, and three-dimensional reconstructed images of rabbits after intravenous injection of 120 mg I / kg iohexol (before injection and 30 min after injection).

[0058] Figure 7 is a transmission electron microscope image of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe from Example 6.

[0059] Figure 8 shows the dynamic light scattering pattern of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe from Example 6.

[0060] Figure 9 shows the relationship between the reciprocal of the longitudinal relaxation time (T1) and the iron ion concentration of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe in Example 6.

[0061] Figure 10 is an abdominal magnetic resonance lymphocyte imaging image of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe in a rabbit model after intramuscular injection.

[0062] Figure 11 is a magnetic resonance imaging of the subcutaneous lymphatic system of the chest after the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe of Example 6 was administered via subcutaneous injection into the axilla of a rabbit model.

[0063] Figure 12 shows the cytotoxicity test results of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe from Example 6.

[0064] Figure 13 shows magnetic resonance (MR) images of comparative example 6-iron-based polydopa amino acid nanomicelles in a rabbit model after intravenous administration using volume rendering (VR) and maximum intensity projection (MIP) techniques; where A: VR-MR images of the abdominal aorta and inferior vena cava; B: MIP-MR images of the abdominal aorta and inferior vena cava; C: MIP-MR images of the abdominal aorta and its branches.

[0065] Figure 14 is a magnetic resonance imaging of the subcutaneous lymphatic system of the chest after the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe of Example 7 was administered via subcutaneous injection into the axilla of a rabbit model.

[0066] Figure 15 is a magnetic resonance imaging of the subcutaneous lymphatic system in the abdomen after the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe of Example 7 was administered via subcutaneous injection in the groin of a rabbit model.

[0067] Figure 16 is a magnetic resonance imaging of the subcutaneous lymphatic system in the abdomen after the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe of Example 8 was administered via subcutaneous injection in the groin of a rabbit model.

[0068] Figure 17 is a magnetic resonance imaging image of the subcutaneous lymphatic system in the abdomen after administration of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe in Example 9 via subcutaneous injection in the groin of a rabbit model. Detailed Implementation

[0069] To enable those skilled in the art to better understand and implement the technical solutions of this application, the following detailed, clear, and complete description of this application is provided in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. Furthermore, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art based on the embodiments of this application without creative effort to obtain all other embodiments should be included within the protection scope of this application.

[0070] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0071] Unless otherwise specified, all raw materials used in this application are commercially available products.

[0072] Example 1: Preparation of polymer nanomicelles.

[0073] The polymer used in this embodiment is poly-DOPA-b-polysarcosine (PDOPA-b-PSar), and its preparation method is an existing technology. It can be synthesized by referring to the polyamino acid preparation method reported in the literature (Miaoer Yu, and Timothy J. Deming, Synthetic Polypeptide Mimics of Marine Adhesives, Macromolecules, 1998, 31(15), 4739-4745) combined with existing copolymerization methods. Alternatively, the amphiphilic polymer containing poly-DOPA amino acids can be prepared by referring to the copolymerization method of dopamine-N-substituted thioanhydride and sarcosine-N-substituted thioanhydride reported in the literature (Yan Qingda. Synthesis of polyamino acids with phenolic hydroxyl groups in the side chain and their application in MRI contrast agents [D], Zhejiang University, 2020).

[0074] First, weigh out 155mg of PDOPA. 37 -b-PSar 118 Dissolve in DMSO, then slowly add a DMSO solution containing 123.3 mg Bi(NO3)3·5H2O. The phenolic hydroxyl group reacts with Bi... 3+ The molar ratio was 3:1. After dialyzing in deionized water for 96 hours, the resulting micelle solution was filtered through a 0.45 μm pore size filter membrane and then brought to a final volume for use.

[0075] The structural formula of the obtained polymer nanomicelles is as follows:

[0076] Example 2: Preparation of polymer nanomicelles.

[0077] This embodiment is basically the same as embodiment 1, except that: PDOPA is used instead of... 37 -b-PSar 118 phenolic hydroxyl group and Bi 3+ The molar ratio is replaced with 2:1.

[0078] Example 3: Preparation of polymer nanomicelles.

[0079] This embodiment is basically the same as Embodiment 1, except that the polymer is replaced with PDOPA. 21 -b-PSar 103 .

[0080] Example 4: Preparation of polymer nanomicelles.

[0081] This embodiment is basically the same as embodiment 3, except that: PDOPA is used instead of... 21 -b-PSar 103 phenolic hydroxyl group and Bi 3+ The molar ratio is replaced with 1:1.

[0082] Example 5: Preparation of polymer nanomicelles.

[0083] This embodiment is basically the same as Embodiment 3, except that the polymer is replaced with PDOPA. 32 -b-PSar 112 .

[0084] I. Particle size and loading efficiency: The average particle size, bismuth ion loading efficiency, and bismuth ion content in the polymer nanomicelles prepared in the examples are shown in Table 1.

[0085] The hydrodynamic diameter of the polymer nanomicelles in solution was detected using a Zetasizer Nano Series (Malvern Instruments) detector at a wavelength of 657 nm and a fixed angle of 90°, with each sample tested in triplicate. The particle size and morphology of the nanomicelles were observed using a HITACHI HT7800 transmission electron microscope with an accelerating voltage of 100 kV.

[0086] The loading efficiency of bismuth ions is calculated as follows: (mass of trivalent bismuth ion compound - mass of remaining trivalent bismuth ion compound) × 100 / mass of trivalent bismuth ion compound.

[0087] The bismuth ion content in polymer nanomicelles is calculated as follows: mass of trivalent bismuth ions × bismuth ion loading efficiency × 100 / (mass of polymer + mass of trivalent bismuth ions × bismuth ion loading efficiency)%.

[0088] Table 1

[0089] The results showed that the polymer nanomicelles prepared in Examples 1-3 had moderate particle size, a trivalent bismuth ion loading efficiency greater than 50%, and a bismuth ion content greater than 15 wt% in the prepared polymer nanomicelles. The TEM image of the polymer nanomicelles is shown in Figure 1; the DLS test results of the polymer nanomicelles are shown in Figure 2.

[0090] Existing technology (Qingda Yan, Xue Dong, et al. Preparation of Mn) 2+ @PolyDOPA-b-polysarcosine micelle as MRI contrast agent with high longitudinal relaxivity[J].Journal of Macromolecular Science,Part APure and Applied Chemistry,2020,DOI:10.1080 / 10601325.2020.1840918) using Mn 2+ The manganese ion content in the polymer nanomicelles prepared by chelation with phenolic hydroxyl groups is approximately 2.5 wt%.

[0091] Existing technology (Jiayu Cen, Botuo Zheng, et al. Ag@polyDOPA-b-polysarcosine hybrid nanoparticles with antimicrobial properties from in-situ reduction and NTA polymerization[J], European Polymer Journal, 2019, DOI:10.1016 / j.eurpolymj.2019.109269) utilizes Ag + The silver ion content in the polymer nanomicelles prepared by chelation with phenolic hydroxyl groups was 5.9 wt%.

[0092] Existing technology (Yuedong Miao, Fengnan Xie, et al. Fe) 3+ @polyDOPA-b-polysarcosine, a T1-Weighted MRI Contrast Agent via Controlled NTA Polymerization[J].ACS Macro Lett.2018,7,693-698,DOI:10.1021 / acsmacrolett.8b00287) The iron ion content in the polymer nanomicelles prepared by chelating Fe3+ with phenolic hydroxyl groups is approximately 9.5 wt%.

[0093] Compared with existing technologies, the present application demonstrates a better chelation effect between trivalent bismuth ions and the catechol structure of polymers, achieving unexpected technical results.

[0094] II. In vitro CT imaging: In vitro CT imaging was measured using a Siemens dual-source CT scanner (SOMATOM Definition Flash, Siemens, Germany) with a tube voltage of 120kV. The results are shown in Figure 3. The CT intensity of the polymer nanomicelles was positively linearly correlated with the bismuth ion concentration, and its CT contrast efficiency was approximately three times that of iohexol used clinically (as shown in Figure 4).

[0095] III. In vivo CT imaging study: The blood pool imaging in rabbits was measured using a Siemens dual-source CT scanner (SOMATOM Definition Flash, Siemens, Germany) with a tube voltage of 120kV. The results are shown in Figure 5. The nanomicelles have excellent cardiovascular imaging effects, and their CT contrast efficiency is significantly better than that of iohexol used in clinical practice (as shown in Figure 6). It can be used as a novel CT contrast agent.

[0096] Example 6: Hyaluronic acid-modified iron-based polydopa amino acid nanoprobe and its preparation.

[0097] The characterization methods involved in this embodiment are as follows: the hydrodynamic particle size of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe was detected on a Zetasizer Nano Series (Malvern Instruments) particle size analyzer; the particle size and morphology of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe were observed and measured on a JEM-1400plus transmission electron microscope; the longitudinal relaxation rate (r1) and magnetic resonance lymphocyte imaging of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe in a rabbit model were detected on a 3.0T magnetic resonance imaging system (SignaHDxt, GE Medical Systems, USA); the cytotoxicity of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe was determined using mouse embryonic fibroblasts (NIH 3T3 cells) by the CCK-8 assay.

[0098] (1) Preparation of hyaluronic acid modified iron-based polydopa amino acid nanoprobes.

[0099] The preparation of polydopa-sarcosine block copolymer was carried out in accordance with the method for preparing polydopa amino acid-polysarcosine block copolymer disclosed in step (1) of Example 6 of Chinese Patent Document CN110496231A.

[0100] The structural formula of the prepared polydopa amino acid-polysarcosine block copolymer is as follows:

[0101] Where R1 is neopentyl, m = 120, n = 25.

[0102] Hyaluronic acid-modified iron-based polydopa amino acid nanoprobes were prepared via a solvent exchange method. A 500 mL round-bottom flask was filled with 4.592 g of polydopa amino acid-polysarcosine block copolymer, followed by 40 mL of DMSO and stirring until dissolved. Ferric nitrate nonahydrate (3.104 g) was dissolved in 20 mL of DMSO and added dropwise to the flask, stirring for 30 min. After stirring, 120 mL of deionized water was slowly added to the flask using a syringe pump, and stirring was continued for 1 h. The mixture was then dialyzed for 24 h (3500 Da, deionized water). After dialysis, an iron-based polydopa amino acid nanomicelle solution with a concentration of 13.28 mg / mL was obtained. Take 10 mL of iron-based polydopa amino acid nanomicelle solution, adjust the pH to 4.0, and slowly add 1.0 mL of hyaluronic acid solution (10 kDa, 10 mg / mL). Stir continuously for 1 h to finally obtain a hyaluronic acid-modified iron-based polydopa amino acid nanoprobe solution (hyaluronic acid mass fraction of 7%). The particle size and morphology of the obtained nanomicelles are shown in Figure 7, with a particle size between 20-30 nm. The hydrodynamic particle size distribution is shown in Figure 8.

[0103] (2) In vitro and in vivo magnetic resonance imaging of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes.

[0104] Hyaluronic acid-modified iron-based polydopa amino acid nanoprobe solutions were serially diluted with different iron ion concentrations. T1-mapping sequences were then used on a 3.0T magnetic resonance imaging (MRI) scanner to determine and calculate the T1 time at different concentrations. The reciprocal of the T1 time was linearly fitted against the iron ion concentration. As shown in Figure 9, the longitudinal relaxation rate of the hyaluronic acid-modified iron-based polydopa amino acid nanoprobe was 4.87 mM. -1 s -1 It is higher than that of commercially available Gd-DTPA magnetic resonance contrast agent.

[0105] Example 7: Preparation of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes.

[0106] In the preparation conditions of the nanoprobe in this embodiment, the amount of hyaluronic acid added was 1 mL (10 kDa, 3 mg / mL), the amount of iron-based polydopa amino acid nanomicelles added was 10 mL (concentration of 13.28 mg / mL), the mass fraction of hyaluronic acid was 2.2%, and other conditions were the same as in Example 6.

[0107] Example 8: Preparation of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes.

[0108] In the preparation conditions of the nanoprobe in this embodiment, the amount of hyaluronic acid added was 0.5 mL (10 kDa, 10 mg / mL), the amount of iron-based polydopa amino acid nanomicelles added was 10 mL (concentration of 10 mg / mL), the mass fraction of hyaluronic acid was 4.8%, and other conditions were the same as in Example 6.

[0109] Example 9: Preparation of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes.

[0110] The structural formula of the prepared polydopa amino acid-polysarcosine block copolymer is as follows:

[0111] Where R1 is neopentyl, m = 131, n = 14.

[0112] In the preparation conditions of the nanoprobe in this embodiment, the amount of hyaluronic acid added was 1 mL (100 kDa, 10 mg / mL), the amount of iron-based polydopa amino acid nanomicelles added was 10 mL (concentration of 15.7 mg / mL), the mass fraction of hyaluronic acid was 6%, and other conditions were the same as in Example 6.

[0113] Comparative Example 1: Iron-based polydopa amino acid nanomicelles.

[0114] The only difference between this embodiment and Embodiment 6 is that hyaluronic acid is not modified on the surface; all other conditions are the same.

[0115] Test Example 1.

[0116] This test case uses a rabbit model to test the magnetic resonance lymphatic imaging effect of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes (from Example 6).

[0117] Healthy New Zealand rabbits weighing 2.0 kg were used. Muscle relaxation was achieved via intramuscular injection of atropine sulfate, followed by anesthesia induced by injection of salbutamol. After successful anesthesia, 0.5 mL of the hyaluronic acid-modified iron-based polydopamine amino acid nanoprobe solution (iron concentration 1 mg / mL) from Example 6 was injected subcutaneously or intramuscularly. Then, 3D imaging of the rabbit's lymphatic vessels and lymph nodes was performed using a fat-suppressed sequence. The New Zealand rabbits before injection served as the pre-group. As shown in Figure 10, after intramuscular injection, significant enhancement of the abdominal lymphatic vessels was observed, clearly showing their structure and course, and the enhancement effect did not diminish after 4 hours. As shown in Figure 11, after subcutaneous injection in the axilla, enhancement of the subcutaneous lymphatic vessels in the chest was observed, with an enhancement time exceeding 4 hours. These results demonstrate that the nanomicelles of this application can serve as a nanocontrast agent for magnetic resonance lymphatic imaging, achieving high-resolution and long-window magnetic resonance imaging of the lymphatic system with simple intramuscular or subcutaneous injection procedures.

[0118] The cytotoxicity of the nanomicelles was determined using the CCK-8 assay on NIH 3T3 cells, with three replicates for each sample. As shown in Figure 12, no significant cytotoxicity was observed in any of the samples at concentrations ranging from 10 to 1000 μg / mL, indicating that the nanomicelles possess good biocompatibility.

[0119] Following the method described above, iron-based polydopa amino acid nanomicelles from Comparative Example 1 were intravenously injected. The abdomen of New Zealand rabbits after tail vein injection was imaged using magnetic resonance (MR) 3D reconstruction with volumetric rendering (VR) and maximum intensity projection (MIP) techniques. The results showed no signal enhancement in the lymphatic system, only significant signal enhancement in the arteries and veins, as shown in Figure 13. A: MR image of the abdominal aorta and inferior vena cava using VR technology (VR-MR); B: MR image of the abdominal aorta and inferior vena cava using MIP technology (MIP-MR); C: MR image of the separated abdominal aorta and its branches using MIP technology (MIP-MR). Enhancement of the abdominal aorta and renal arteries / veins was observed, while no significant enhancement was seen in the abdominal lymphatic vessels, indicating that the iron-based polydopa amino acid nanomicelles from Comparative Example 1 could not leak from the blood vessels.

[0120] Test Example 2.

[0121] This test case uses a rabbit model to test the magnetic resonance lymphometry (MRL) effect of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes (from Example 7). The test conditions, including the animal model, MR scanning sequence, and animal experimental procedure, are consistent with those in Example 6. As shown in Figure 14, after subcutaneous injection in the axilla, enhancement of the axillary lymph nodes was observed, lasting for more than 6 hours and disappearing after 24 hours. As shown in Figure 15, after subcutaneous injection in the groin, significant enhancement of the abdominal lymphatic vessels was observed, lasting for more than 6 hours and disappearing after 24 hours as well.

[0122] Test Example 3.

[0123] This test case uses a rabbit model to test the magnetic resonance lymphatic imaging effect of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes (from Example 8). The test conditions, including the animal model, magnetic resonance scanning sequence, and animal experimental procedure, are consistent with those in Example 6. As shown in Figure 16, after subcutaneous injection in the right groin, significant enhancement of abdominal lymphatic vessels was observed, with the enhancement time exceeding 3 hours. The direction of abdominal lymphatic vessels and lymph nodes could be seen (red arrows). The left side, without nanoprobe injection, served as a control (green arrows).

[0124] Test Example 4.

[0125] This test case used a rabbit model to evaluate the magnetic resonance lymphometry (MRL) effect of hyaluronic acid-modified iron-based polydopa amino acid nanoprobes (from Example 9). The test conditions, including the animal model, MR scanning sequence, and animal experimental procedure, were consistent with those in Example 6. As shown in Figure 17, after subcutaneous injection in the right groin, enhanced abdominal lymph nodes were observed for more than 1 hour (blue arrow). After 24 hours, the nanoprobes accumulated in the lymph nodes, causing significant enhanced MR signals (red arrow).

[0126] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An amphiphilic block copolymer comprising hydrophobic blocks and hydrophilic blocks; characterized in that, The hydrophobic block is a polydopa amino acid, and its repeating unit length is any integer between 1 and 200; the hydrophilic block is selected from at least one of polysarcosine, polyethylene glycol, polyvinylpyrrolidone, polyacrylic acid, polyacrylamide, poly-N-ethylglycine, polyglutamic acid, polymethyl oxazoline, polyethyl oxazoline, monomethyl ether polyethylene glycol, polyethylene oxide, or polyvinyl alcohol, and its structural unit number is any integer between 1 and 2000.

2. The amphiphilic block copolymer according to claim 1, characterized in that, The topological structures of the amphiphilic block copolymers include diblock, triblock, multiblock, random, star, ring, or grafted structures.

3. The amphiphilic block copolymer according to claim 1, characterized in that, The structure of the polymer is shown in formula (1) and / or formula (2): In formulas (1) and (2), R1 is independently selected from alkyl, benzyl, or silyl groups; m is the number of hydrophilic block structural units, which can be any integer between 1 and 2000; n is the length of the polydopa amino acid chain segment, which can be any integer between 1 and 200.

4. A polymer nanomicelle, characterized in that, The polymer nanomicelles are chelates of trivalent bismuth ions chelated by the phenolic hydroxyl groups of the polymer side chains, and the polymer is an amphiphilic block copolymer as described in any one of claims 1 to 3. The structure of the chelate is as follows: The molar ratio of phenolic hydroxyl groups to trivalent bismuth ions in the polymer nanomicelles is 3:1 to 2; 5. The polymer nanomicelles according to claim 4, characterized in that, The trivalent bismuth ion compound is selected from at least one of bismuth nitrate, bismuth chloride, bismuth sulfate, and bismuth ammonium citrate.

6. The polymer nanomicelles according to claim 4, characterized in that, The polymer nanomicelles have a particle size of 10–500 nm.

7. The polymer nanomicelles according to claim 4, characterized in that, The polymer nanomicelles have a bismuth ion loading efficiency of ≥50% and a bismuth ion content of ≥15wt%.

8. The polymer nanomicelles according to claim 4, characterized in that, The connection length of the hydrophilic block is 50 to 200, and the connection length of the hydrophobic block is 1 to 50.

9. The use of a polymer nanomicelle according to any one of claims 4 to 8 in the preparation of a computed tomography imaging agent.

10. A CT contrast agent, characterized in that, It includes the polymer nanomicelles according to any one of claims 1 to 8, and medically or pharmaceutically acceptable excipients, including but not limited to excipients and diluents.

11. A method for preparing polymer nanomicelles according to any one of claims 4 to 8, characterized in that: Includes the following steps: (1) Preparation of amphiphilic block copolymers according to any one of claims 1 to 3: polydopa block-hydrophilic block copolymers are obtained by ring-opening polymerization of N-substituted thioanhydrides with dopa amino acid monomers and hydrophilic monomers. (2) Preparation of CT imaging nanomicelles by solvent displacement method: The copolymer obtained in step (1) is dissolved in DMSO, and the trivalent bismuth ion compound of claim 5 is added, wherein the molar ratio of phenolic hydroxyl group to trivalent bismuth ion is 3:1 to 2. After dialyzing with deionized water, the mixture is filtered and the volume is adjusted to obtain the polymer nanomicelles.

12. A polymer nanomicelle according to any one of claims 4 to 8, characterized in that, The trivalent bismuth ion can be replaced by at least one of tantalum ion, tungsten ion, gadolinium ion, holmium ion, ytterbium ion, lutetium ion, yttrium ion, rhenium ion, actinium ion, and gold ion.

13. A hyaluronic acid-modified iron-based polymer nanomicelle probe, characterized in that, It includes iron-based nanomicelles and hyaluronic acid modified on their outer layer; the iron-based nanomicelles are obtained by chelating the amphiphilic block copolymer according to any one of claims 1 to 3 with a trivalent iron ion compound; Hyaluronic acid was modified on the outer layer of iron-based nanomicelles via solvent exchange. The mass ratio of hyaluronic acid to iron-based nanomicelles is 2–10:90–98, and the molecular weight of hyaluronic acid is 1–1000 kDa.

14. The hyaluronic acid-modified iron-based polymer nanomicelle probe according to claim 13, characterized in that, The iron-based nanomicelles are spherical, cylindrical, or vesicle-shaped assemblies; the amphiphilic block copolymers have linear, star-shaped, grafted, or hyperbranched structures; the hydrophilic blocks have a connection length of 50–200, and the hydrophobic blocks have a connection length of 1–50.

15. The hyaluronic acid-modified iron-based polymer nanomicelle probe according to claim 13, characterized in that, The iron-based nanomicelles are neutral to slightly negatively charged. The iron-based nanomicelles are connected to hyaluronic acid through multiple hydrogen bond interactions between the polyhydroxyl groups of hyaluronic acid (hydrogen bond donors) and the polyamino acid amide bonds (hydrogen bond acceptors) of the iron-based nanomicelle shell.

16. The hyaluronic acid-modified iron-based polymer nanomicelle probe according to claim 13, characterized in that, The hyaluronic acid-modified iron-based polydopa amino acid nanoprobe has a particle size of 6–500 nm and an iron content of 10–1000 ppm.

17. The application of a hyaluronic acid-modified iron-based polymer nanomicelle probe according to any one of claims 13 to 16 in the preparation of a T1-enhanced magnetic resonance lymphatic imaging contrast agent, wherein the administration method of the application includes at least one of subcutaneous injection, intramuscular injection, and intralymphatic injection.

18. The application of a hyaluronic acid-modified iron-based polymer nanomicelle probe according to any one of claims 13 to 16 in the imaging of targeted lymphatic vessels and targeted lymph nodes.

19. A method for preparing a hyaluronic acid-modified iron-based polymer nanomicelle probe according to any one of claims 13 to 16, characterized in that: Includes the following steps: (1) Preparation of the amphiphilic block copolymer according to claim 1: polydopa block-hydrophilic block copolymer is obtained by ring-opening polymerization of N-substituted thioanhydride with dopa amino acid monomer and hydrophilic monomer. (2) Preparation of nanoprobes for magnetic resonance lymphocyte imaging: (2.1) The copolymer obtained in step (1) is added to DMSO and stirred until dissolved; (2.2) Add ferric ion compound, stir and dialyze with deionized water for 24 h to obtain iron-based nano micelle solution; (2.3) Adjust the pH of the iron-based nanomicelle solution to 4.0-5.0, slowly add hyaluronic acid solution, and stir continuously to finally obtain a nanoprobe for magnetic resonance lymphoma imaging.

20. The hyaluronic acid-modified iron-based polymer nanomicelle probe according to claim 13, characterized in that, The trivalent iron ion can be replaced by at least one of tantalum ion, tungsten ion, gadolinium ion, holmium ion, ytterbium ion, lutetium ion, yttrium ion, rhenium ion, actinium ion, and gold ion.

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