Developing agent and preparation method thereof, nonionic developing microsphere and preparation method and application thereof

Abstract

The invention provides a developing agent which is modified iopamidol, a clinically safe nonionic hydrophilic developing agent iopamidol is subjected to structural modification, a hydrophilic side chain and a connecting arm are introduced, and the iopamidol is introduced into a gelatin degradable microsphere matrix with good biocompatibility in a covalent bonding manner; the degradable non-ionic X-ray developing microsphere with good biocompatibility is formed. Developing microspheres prepared by adopting the developing agent provided by the invention not only keep excellent X-ray developing performance, solve the problems of aggregation tendency and biocompatibility of traditional developing microspheres and realize real-time visual monitoring of an embolism process, but also have degradability and good biological safety, and are not only suitable for permanent embolism treatment, but also suitable for treatment of embolism. The solid developing embolism agent can also be expanded to the fields of temporary embolism, tissue engineering and the like, and the solid developing embolism agent with precise developing and safe degradation performance is provided for minimally invasive interventional therapy of diseases such as tumor intervention and vascular malformation.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a non-ionic radiopaque microsphere, its preparation method, and its application. Background Technology

[0002] Transcatheter arterial embolization (TAE) is a minimally invasive interventional treatment method that involves inserting a microcatheter into a target blood vessel through a percutaneous approach and precisely delivering an embolic agent to block blood flow. It is widely used in clinical settings such as hypervascular tumors like liver cancer, kidney cancer, uterine fibroids, cerebral vascular malformations, and acute bleeding in internal organs. Its minimally invasive nature significantly reduces patient trauma, shortens recovery time, and lowers the risk of surgical complications, playing a crucial role, especially in the treatment of emergency massive hemorrhage. The choice of embolic agent is critical to treatment efficacy and patient safety. Among these, embolic microspheres, as solid embolic agents with high operability and controllability, have attracted considerable attention. Based on functional characteristics, they can be classified into blank embolic microspheres, drug-loaded or radionuclide-loaded embolic microspheres, and contrast-enhanced embolic microspheres. Contrast-enhanced embolic microspheres, due to the introduction of contrast agents or materials, facilitate intraoperative and postoperative imaging monitoring, making them an important research direction for improving the precision and safety of interventional treatments.

[0003] Currently, the blank embolization microspheres and drug-loaded embolization microspheres commonly used in clinical practice do not possess X-ray imaging properties. They typically need to be mixed with contrast agents to achieve temporary intraoperative imaging through physical adsorption of the contrast agent. However, because the contrast agent easily diffuses out of the embolization microspheres, commonly used X-ray imaging techniques such as computed tomography (CT) and digital subtraction angiography (DSA) struggle to provide postoperative follow-up evaluation of the embolization agent. To endow embolization microspheres with stable imaging capabilities, existing technologies mainly employ two strategies: physical modification and chemical modification. Physical modification methods mainly involve encapsulating X-ray imaging nanoparticles such as barium sulfate, gold, liquid metal, or tantalum into embolization microspheres. However, due to uneven distribution of nanoparticles, limited concentration, and potential leakage risks, the imaging effect is difficult to meet clinical requirements. Chemical modification strategies involve covalently bonding imaging groups to the polymer chain of microspheres. Among them, triiodobenzene compounds are the most important core groups of iodine-containing contrast agents in the field of medical imaging. Their molecular structure is based on benzene rings, and they achieve excellent X-ray absorption capabilities through the high electron density of three iodine atoms. For example, the LC Bead LUMI imaging blank embolization microspheres and DC Bead LUMI imaging drug-loaded embolization microspheres developed by Boston Scientific in 2016, and the Vispearl imaging drug-loaded embolization microspheres launched by Huihe Medical in China in 2024, all achieve imaging by chemically modifying polyvinyl alcohol with triiodobenzene. However, the strong hydrophobicity and high density of triiodophenyl groups present a series of technical challenges: In terms of synthesis, their high hydrophobicity necessitates complex processes such as critical phase surface crosslinking to ensure uniform distribution of radioactive groups and hydrophilicity of the microsphere surface. This not only increases production costs but also affects process stability and repeatability. In clinical applications, the high density of triiodophenyl groups increases the overall density and rigidity of the microspheres, making microcatheter delivery difficult and affecting the safety and precision of surgical procedures. More importantly, the potential cytotoxicity of triiodophenyl groups limits their application in biodegradable embolization microspheres, currently confining them mainly to non-degradable material systems and hindering their expansion into emerging application areas such as tissue engineering and temporary embolization.

[0004] In summary, among existing contrast-enhanced embolization microsphere technologies, physical modification methods are insufficient to meet clinical needs due to issues with contrast stability and safety. Traditional chemical modification methods are limited by the hydrophobicity, high density, and potential toxicity of the triiodophenyl group, resulting in significant shortcomings in terms of process cost, operational performance, and application scope. Meanwhile, contrast-enhancing methods relying on physical adsorption of contrast agents cannot achieve effective postoperative monitoring. The current technology system has not yet been able to simultaneously consider contrast performance, biocompatibility, process feasibility, and clinical ease of operation. In particular, there is a lack of biodegradable contrast-enhanced embolization microsphere products, making it difficult to meet the needs of precision treatment in the diagnosis and treatment of diseases such as interventional tumors and vascular malformations.

[0005] Therefore, providing a radiopaque microsphere product that can simultaneously take into account radiopaque performance, biocompatibility, process feasibility, and ease of clinical operation, especially with biodegradable properties, has become a problem that needs to be solved. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a developing agent and its preparation method, a non-ionic developing microsphere and its preparation method and application. The developing microsphere product prepared by the developing agent provided by the present invention can simultaneously take into account developing performance, biocompatibility, process feasibility and clinical operation convenience.

[0007] This invention provides a developer, wherein the developer is a modified iopamidol, and the modified iopamidol has the structure shown in Formula IV:

[0008] Formula IV

[0009] In equation IV, R 2 Selected from at least one group having the following structure:

[0010]

[0011] n is any integer greater than or equal to 1;

[0012] R 3 Selected from at least one group having the following structure:

[0013] .

[0014] Preferably, the developer is selected from at least one compound having the following structure:

[0015] .

[0016] The present invention also provides a method for preparing the above-mentioned developer, comprising the following steps:

[0017] Iopamidol with protected hydroxyl groups was condensed with a carboxylic acid compound and then deprotected to obtain a developer.

[0018] The filamentous amino alcohol hydroxyl group protected by iopamidol has the structure shown in Formula I:

[0019] Formula I

[0020] The R 1 Independently selected from groups having the following structures:

[0021]

[0022]

[0023] Or, R 1 The oxygen atoms connected to them form a ring, and the ring has any of the following structures:

[0024]

[0025] ;

[0026] The carboxylic acid compound has the structure shown in Formula II:

[0027] Formula II

[0028] In Equation II, R 2 Selected from at least one group having the following structure:

[0029]

[0030] n is any integer greater than or equal to 1;

[0031] R 3 Selected from at least one group having the following structure:

[0032] .

[0033] Preferably, the condensation reaction is carried out under conditions of a catalyst and an activator, wherein some condensation conditions require the addition of a base to replace the catalyst;

[0034] The catalyst is selected from 4-dimethylaminopyridine;

[0035] The base is selected from at least one of N,N-diisopropylethylamine, triethylamine, N-methyldiisopropylamine, pyridine, and N-methylmorpholine.

[0036] The activator is selected from carboxyl activators, and the carboxyl activator is selected from at least one of the following: dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, cyclohexylisobutylcarbodiimide, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate.

[0037] The deprotection reagent used is selected from tetrabutylammonium fluoride, hydrochloric acid, and sodium hydroxide.

[0038] The present invention also provides a nonionic developing microsphere, which is prepared from a developing agent and a biomaterial matrix, wherein the developing agent is selected from the above-mentioned developing agents.

[0039] Preferably, the biomaterial matrix is ​​selected from one or more of natural biodegradable polymers and / or synthetic polymers;

[0040] The natural biodegradable polymer is selected from gelatin, chitosan, sodium hyaluronate, sodium alginate, and carrageenan;

[0041] The synthetic polymer is selected from polylactic acid-hydroxyacetic acid copolymer, polycaprolactone, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, and polyacrylamide.

[0042] Preferably, the gelatin is selected from at least one of unmodified gelatin and modified gelatin;

[0043] The modification is as follows:

[0044] Modify the -NH2 group in the gelatin structure with any of the following groups:

[0045] , , ;

[0046] n is any integer greater than or equal to 1;

[0047] And / or, modify the -COOH group in the gelatin structure with the following groups:

[0048] .

[0049] Preferably, in the nonionic developing microspheres, the loading of the developer is 5-20 wt%;

[0050] The biomaterial matrix is ​​selected from gelatin, and the degree of cross-linking of the gelatin is 30%-70%.

[0051] Preferably, the non-ionic developing microspheres have an ellipsoidal structure, with a minor axis diameter of 100-700 μm, a length of 500-1200 μm, and a density of 1.05-1.3 g / cm³. 3 .

[0052] Preferably, the non-ionic radiopaque microspheres further include a drug, which is loaded in the biomaterial matrix.

[0053] The present invention also provides a method for preparing the above-mentioned nonionic radiopaque microspheres, comprising the following steps:

[0054] A) The developer is cross-linked with the biomaterial matrix using a microfluidic method to obtain microspheres, which are non-ionic developer microspheres;

[0055] B) The microspheres are post-processed to obtain non-ionic radiopaque microspheres that can be used for in vivo experiments.

[0056] Preferably, the crosslinking includes at least one of ultraviolet light crosslinking and chemical crosslinking.

[0057] Preferably, step A) includes:

[0058] Using a microfluidic chip, the continuous phase and the dispersed phase are intersected and sheared in a microchannel. Under ultraviolet light irradiation, the dispersed phase droplets formed by shearing undergo a cross-linking reaction to obtain microsphere particles.

[0059] The continuous phase is selected from at least one of soybean oil and mineral oil;

[0060] The dispersed phase comprises an aqueous solution of modified iopamidol developer, biomaterial matrix, photoinitiator, and PBS buffer (pH 7.4);

[0061] The photoinitiator includes, but is not limited to, at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and 1-hydroxycyclohexylphenyl ketone;

[0062] The flow rate of the continuous phase is 20-30 μL / min, and the flow rate of the dispersed phase is 2-12 μL / min;

[0063] The cross-linking reaction is carried out at a temperature of 40~90℃.

[0064] The present invention also provides the application of the above-mentioned non-ionic radiopaque microspheres in the preparation of radiotherapy, chemotherapy and embolization combined therapy drugs and radiopaque diagnostic reagents for tumors.

[0065] Compared with existing technologies, this invention provides a contrast agent, namely modified iopamidol. This invention modifies the structure of iopamidol, a clinically safe nonionic hydrophilic contrast agent, by introducing hydrophilic side chains and linkers, and covalently incorporating it into a biocompatible gelatin biodegradable microsphere matrix to form a biocompatible biodegradable nonionic X-ray contrast microsphere. In this invention, the original hydrophilic serine amino alcohol structure of iopamidol and the newly introduced hydrophilic side chains effectively improve the hydrophobicity of the contrast microspheres; the diffusion of the contrast agent is prevented by covalently bonding the linkers on the modified iopamidol to the groups on the gelatin. The contrast-enhancing microspheres prepared using the contrast agent provided by this invention retain excellent X-ray imaging performance, solve the aggregation tendency and biocompatibility problems of traditional contrast-enhancing microspheres, and realize real-time visual monitoring of the embolization process. They also have degradability and good biosafety, making them suitable not only for permanent embolization treatment but also for temporary embolization, tissue engineering, and other fields. They provide a solid contrast-enhancing embolization agent with both precise imaging and safe degradation performance for minimally invasive interventional treatment of diseases such as tumor intervention and vascular malformations, effectively overcoming the shortcomings of existing technologies such as easy diffusion of contrast agents, unstable imaging effect, complex process, poor biocompatibility, and limited application scenarios. Attached Figure Description

[0066] Figure 1 An optical microscope image of the non-ionic microspheres prepared in Example 1;

[0067] Figure 2 X-ray imaging of the non-ionic microspheres prepared in Example 1;

[0068] Figure 3 The image shows a micro-computed tomography (CT) image of the non-ionic imaging microspheres prepared in Example 1.

[0069] Figure 4 The image shows an optical microscope image of the non-ionic microspheres prepared in Example 2.

[0070] Figure 5 X-ray imaging of the non-ionic microspheres prepared in Example 2;

[0071] Figure 6 The image shows a micro-computed tomography (CT) image of the non-ionic imaging microspheres prepared in Example 2.

[0072] Figure 7 The image shows an optical microscope image of the non-ionic microspheres prepared in Example 3.

[0073] Figure 8 X-ray imaging of the non-ionic microspheres prepared in Example 3;

[0074] Figure 9 The image shows a micro-computed tomography (CT) image of the non-ionic imaging microspheres prepared in Example 3.

[0075] Figure 10 The image shows the degradation and CT imaging changes of the non-ionic radiopaque microspheres prepared in Example 3 in the ears of New Zealand rabbits. Detailed Implementation

[0076] This invention provides a developer, wherein the developer is a modified iopamidol, and the modified iopamidol has the structure shown in Formula IV:

[0077] Formula IV

[0078] In equation IV, R 2 Selected from at least one group having the following structure:

[0079]

[0080] n is any integer greater than or equal to 1, preferably any integer between 1 and 10, and more preferably any integer between 1 and 5;

[0081] R 3 Selected from at least one group having the following structure:

[0082] .

[0083] In some specific embodiments of the present invention, the developer is selected from at least one of compounds having the following structures:

[0084] .

[0085] The modified iopamidol provided by this invention retains the triiodobenzene developing core structure of iopamidol, and enhances its hydrophilicity by connecting hydrophilic side chains (selected from PEG, PVA, PAA, etc.) via ester bonds. It also contains alkenyl, glycidyl, and alkynyl linker functional groups. The modified iopamidol molecule is nonionic, possessing both high iodine content developing ability and good water solubility, and exhibits excellent biocompatibility.

[0086] The present invention also provides a method for preparing a developer, comprising the following steps:

[0087] Iopamidol with protected hydroxyl groups was condensed with a carboxylic acid compound and then deprotected to obtain a developer.

[0088] The filamentous amino alcohol hydroxyl group protected by iopamidol has the structure shown in Formula I:

[0089] Formula I

[0090] The R 1 Independently selected from groups having the following structures:

[0091]

[0092]

[0093] Or, R 1 The oxygen atoms connected to them form a ring, and the ring has any of the following structures:

[0094]

[0095] ;

[0096] The carboxylic acid compound has the structure shown in Formula II:

[0097] Formula II

[0098] In Equation II, R 2 Selected from at least one group having the following structure:

[0099]

[0100] n is any integer greater than or equal to 1;

[0101] R 3 Selected from at least one group having the following structure:

[0102] .

[0103] Specifically, iopamidol (Formula I), with its protected hydroxyl groups, is used as a reactant. The exposed hydroxyl groups in iopamidol (Formula I) react with hydrophilic side chains (R... 2 ) and connecting arm (R 3 The carboxylic acid compound (Formula II) undergoes a condensation reaction to yield the intermediate iopamidol (Formula III). Further removal of the protecting group from iopamidol (Formula III) yields modified iopamidol (Formula IV). The specific chemical reaction formulas are as follows:

[0104]

[0105] In the synthetic route of this invention, iopamidol (Formula I) with protected hydroxyl groups of filament amino alcohol is used as the starting material. This choice of starting material not only preserves the triiodobenzene structure in the iopamidol molecule as the development core, but also avoids unnecessary side reactions during the reaction process through the hydroxyl protection strategy, thus ensuring the site specificity of molecular modification.

[0106] In the iopamidol with its hydroxyl groups protected, cyclic protecting groups can be formed with hydroxyl groups. These mainly include acetal structures formed by condensation with formaldehyde, acetaldehyde, and benzaldehyde, ketal structures formed by condensation with acetone, and borate ester protecting groups formed by condensation with methylboronic acid, ethoxyboronic acid, or phenylboronic acid.

[0107] The ring has any of the following structures:

[0108]

[0109]

[0110] In the iopamidol with its protected hydroxyl groups, the hydroxyl protecting groups that can be used mainly include acyl, alkyl, ester, and silyl protecting groups. Acyl protecting groups mainly include acetyl (Ac), propionyl (Pr), pivaloyl (Piv), and benzoyl (Bz); alkyl protecting groups mainly include methyl (Me), tert-butyl (tBu), benzyl (Bn), and MOM protecting groups; ester protecting groups include methoxycarbonyl (COOMe) and tert-butoxycarbonyl (Boc); and silyl protecting groups include trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and tert-butyldiphenylsilyl (TBDPS). The protecting groups have the following structures:

[0111]

[0112] .

[0113] During the condensation reaction stage, the carboxylic acid compound having the structure of Formula II undergoes a condensation reaction with the exposed hydroxyl group in iopamidol having the protected hydroxyl group of the serine amino alcohol having the structure of Formula I via an activated carboxyl group to generate an ester group. This carboxylic acid molecule also possesses a side chain (R 2 ) and connecting arm (R 3 ) structure. Among them, the side chain (R 2 R is a key structure for improving the hydrophilicity of imaging microspheres and reducing non-specific adsorption to proteins in the blood. 2 The selected structures mainly include linear alkyl groups, polyoxymethylene (POM), polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(acrylate) (PAA), sodium polyacrylate (SPA), polyacrylamide (PAM), and poly(N,N-dimethylacrylamide) (PDMAA). Specifically, they have the following structures:

[0114]

[0115] n is any integer greater than or equal to 1, preferably any integer between 1 and 10, and more preferably any integer between 1 and 5;

[0116] In carboxylic acid compounds having the structure of formula II, the connecting arm R 3 The key to achieving the binding of modified iopamidol to the gelatin backbone lies in its chemical structure, which can specifically match the active groups such as amino, hydroxyl, and carboxyl groups in the gelatin molecule, mainly including alkenyl, propylene oxide, alkynyl, azide, aldehyde, carboxyl, and maleimide groups. 3 Selected from at least one group having the following structure:

[0117] .

[0118] The condensation reaction is carried out under the conditions of a catalyst and an activator, wherein some condensation reactions require the addition of a base to replace the catalyst;

[0119] The catalyst is selected from 4-dimethylaminopyridine (DMAP);

[0120] The base is selected from at least one of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), N-methyldiisopropylamine (DMEA), pyridine, and N-methylmorpholine (NMM);

[0121] The activator is selected from carboxyl activators, and the carboxyl activator is selected from at least one of the following: dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), cyclohexylisobutylcarbodiimide (CIC), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU).

[0122] The deprotection reagent used is selected from tetrabutylammonium fluoride (TBAF), hydrochloric acid, and sodium hydroxide.

[0123] Finally, after deprotection, the developer is obtained through separation and purification.

[0124] The present invention also provides a nonionic developing microsphere, which is prepared from a developing agent and a biomaterial matrix, wherein the developing agent is selected from the developing agents described above.

[0125] The biomaterial matrix is ​​selected from one or more of natural biodegradable polymers and / or synthetic polymers.

[0126] The natural biodegradable polymers are selected from gelatin, chitosan, sodium hyaluronate, sodium alginate, and carrageenan. Gelatin molecules contain active groups such as amino, hydroxyl, and carboxyl groups, which can covalently link with the contrast agent. Furthermore, the gelatin structure can be further modified to introduce new linking sites, facilitating covalent bonding with other structures. Chitosan contains amino groups, which can react with the carboxyl / activated olefin groups of the contrast agent; its slower degradation makes it suitable for medium- to long-term embolization. Sodium hyaluronate contains hydroxyl and carboxyl groups, exhibiting strong hydrophilicity and improving blood compatibility. Sodium alginate binds to the contrast agent through uronic acid groups, and its mechanical properties can be controlled through calcium ion-assisted molding. Carrageenan contains hydroxyl and sulfate ester groups, and can be grafted with contrast agents through nucleophilic substitution or esterification reactions. Therefore, all of the above are covalently linked to the modified contrast agent through the active groups of the natural biodegradable polymer itself or through sites introduced by modification.

[0127] The synthetic polymer is selected from polylactic-co-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyacrylic acid, and polyacrylamide (PAM). PLGA contains terminal hydroxyl and carboxyl groups and is grafted via esterification or amidation. PCL contains ester and terminal hydroxyl groups and is combined with a developer via transesterification or condensation. This synthetic polymer maintains development stability and biodegradability while allowing for flexible adjustment of the microsphere degradation cycle and mechanical properties to ensure injectability via microcatheters.

[0128] In this invention, the biomaterial matrix is ​​selected from gelatin, and the gelatin is selected from at least one of unmodified gelatin and modified gelatin;

[0129] The modification is as follows:

[0130] Modify the -NH2 group in the gelatin structure with any of the following groups:

[0131] , , ;

[0132] And / or, modify the -COOH group in the gelatin structure with the following groups:

[0133] .

[0134] This invention modifies gelatin to introduce new bonding sites, making it easier to covalently bond with other structures.

[0135] Specifically, the amino groups on gelatin act as nucleophiles and undergo an amidation reaction with methacrylic anhydride. The methacrylic acid structural units are covalently linked to the gelatin backbone via stable amide bonds, thereby introducing olefin double bond linkage sites into the gelatin. The chemical reaction formula is as follows:

[0136]

[0137] The amino groups on gelatin can also undergo amidation reactions with azido-N-hydroxysuccinimide esters of various alkyl lengths, introducing azido groups into the gelatin backbone that are physiologically inert and hardly react with any biomolecules. The chemical reaction formula is as follows:

[0138]

[0139] The nucleophilicity of the amino groups on gelatin attacks 2-iminothione (Traut's reagent) molecules, causing a ring-opening reaction to obtain gelatin containing thiol-linked sites. The chemical reaction formula is as follows:

[0140]

[0141] The carboxyl groups on gelatin are also a region where new linkage sites can be easily introduced. A strategy of activating the carboxyl groups via EDC / NHS, followed by a reaction with mercaptoethylamine, introduces terminal thiol groups into the gelatin backbone. The chemical reaction formula is as follows:

[0142]

[0143] In the nonionic developing microspheres, the loading of the developer is 5-20 wt%, which can be 5 wt%, 7 wt%, 10 wt%, 12 wt%, 15 wt%, 17 wt%, 20 wt%, or any value between 5-20 wt%.

[0144] The biomaterial matrix is ​​selected from gelatin, and the degree of crosslinking of the gelatin is 30%-70%, which can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or any value between 30% and 70%.

[0145] Based on clinical needs (such as 6-12 months of tumor treatment, 1-3 months of temporary embolization), this invention ensures that the CT / DSA imaging clarity of the microspheres throughout the degradation process meets monitoring requirements by controlling the loading of the contrast agent and the degree of cross-linking of the gelatin, thereby achieving precise matching between imaging time and treatment cycle.

[0146] This invention uses gelatin as a three-dimensional network cross-linked framework, with modified iopamidol uniformly dispersed within it through covalent bonds. The iodine atom distribution density and the degree of cross-linking of the microspheres are controllable. The microspheres are capsule-shaped, with uniform particle size and smooth surface. They possess both a density of 1.05-1.3 g / cm³ and suitable mechanical properties. The gelatin matrix provides them with a degradation cycle of 1-12 months, and their development performance remains stable during the degradation process.

[0147] The non-ionic developing microspheres have a near-ellipsoidal structure. The diameter of the non-ionic developing microspheres ranges from 100 to 700 μm (minor axis diameter), and can be any value of 100, 200, 300, 400, 500, 600, 700, or 100-700 μm; the length is 500-1200 μm, and can be any value between 500, 600, 700, 800, 900, 1000, 1100, 1200, or 500-1200 μm; the density is 1.05-1.3 g / cm³. 3 It can be 1.05, 1.1, 1.15, 1.2, 1.3, or 1.05-1.3 g / cm³. 3 Any value between; the degradation cycle is 1 to 12 months.

[0148] In existing technologies, it is difficult to balance the content of the contrast agent with the mechanical properties of the microspheres. For example, excessively high triiodophenyl group density leads to increased rigidity of the microspheres, making delivery difficult. This invention, by adjusting the amount and type of modified iopamidol added, can quantitatively control the iodine atom density (development effect) and mechanical properties (such as rigidity and elasticity) in the microspheres. At the same time, it combines microfluidic technology to precisely control the size of the microspheres (aspect ratio and minor diameter), balancing imaging clarity and clinical ease of operation.

[0149] In existing technologies, the hydrophobicity of imaging microspheres easily leads to non-specific adsorption of blood components, affecting biocompatibility. This invention addresses this by designing functionalized side chains. Through the synergistic effect of the high hydration properties of hydrophilic groups, such as polyethylene glycol, and the hydrophilic side chains of iopamidol itself, the hydrophobicity of the microspheres is reduced, the tendency to aggregate is decreased, and biocompatibility is improved.

[0150] The present invention provides biodegradable contrast-enhancing microspheres for X-ray visualization in vascular interventions, which are absorbable by the human body. During surgery, X-ray visualization helps doctors achieve precise vascular embolization. Secondly, the contrast agent molecules in the microspheres are covalently bound to the biomaterial matrix, allowing their contrast ability to change linearly with the matrix degradation process. This characteristic enables real-time monitoring and assessment of the microspheres' degradation in vivo via non-invasive CT scans during subsequent treatment, thus providing a valid basis for subsequent medical decisions.

[0151] The present invention also provides a method for preparing the above-mentioned nonionic radiopaque microspheres, comprising the following steps:

[0152] A) The developer is cross-linked with the biomaterial matrix using a microfluidic method to obtain microspheres, which are non-ionic developer microspheres;

[0153] B) The microspheres are post-processed, cleaned, and disinfected to obtain non-ionic radioactive microspheres suitable for in vivo experiments.

[0154] First, microfluidic chips are used in conjunction with photolithography to fabricate flow channel structures with different sizes and specifications. The precise design of these flow channel structures is the foundation for the subsequent effective control of microsphere size, and can provide a stable and controllable spatial environment for the formation of microspheres.

[0155] By adjusting the flow rate ratio of the microsyringe, the formation process of microspheres in the flow channel can be precisely controlled, thereby preparing capsule-shaped embolization microspheres with different aspect ratios and minor diameters to meet the morphological requirements of microspheres in different clinical applications.

[0156] In this invention, the flow rate of the continuous phase is 20-30 μL / min, which can be any value between 20, 22, 24, 25, 26, 28, 30, or 20-30 μL / min, and the flow rate of the dispersed phase is 2-12 μL / min, which can be any value between 2, 4, 5, 6, 8, 10, 12, or 2-12 μL / min.

[0157] In this invention, the biomaterial matrix is ​​preferably gelatin, and the gelatin is selected from modified gelatin and / or unmodified gelatin. Since both modified iopamidol and gelatin possess a variety of selectable active binding sites, diverse UV crosslinking or chemical crosslinking strategies can be employed to covalently link modified iopamidol and gelatin.

[0158] Specifically, it mainly includes the following types of chemical reactions:

[0159] Under heating conditions, the amino groups on gelatin and the propylene oxide on modified iopamidol undergo a nucleophilic ring-opening reaction to form CN bonds, achieving covalent bonding between the two. At the same time, the newly generated hydroxyl groups further enhance the hydrophilicity of gelatin.

[0160]

[0161] The hydroxyl groups on gelatin undergo a nucleophilic ring-opening reaction with the propylene oxide on modified iopamidol to form a hydroxypropyl ether structure. This reaction is competitive, meaning the amino groups on gelatin are far more reactive than the hydroxyl groups, resulting in low chemoselectivity for the direct reaction. Therefore, the reaction needs to be adjusted to a high pH condition. Under high pH conditions, the hydroxyl groups partially deprotonate, generating more nucleophilic alkoxides, thereby increasing the reactivity of the hydroxyl groups.

[0162]

[0163] When thiol groups are introduced into gelatin, the amino group is partially protonated when the pH of the reaction system is about 5.0-7.0, resulting in the thiol group having higher reactivity than the amino group. Therefore, the thiol group reaction site can easily undergo a ring-opening reaction with propylene oxide to form a hydroxypropyl sulfide structure.

[0164]

[0165] The high reactivity of the thiol group also allows it to undergo a Michael addition reaction with maleamide to form a thioether bond, thus achieving covalent linkage.

[0166]

[0167] The thiol groups on gelatin can also react with the olefin double bonds on iopamidol in a photo-initiated thiol-olefin click reaction, which can also form thioether bonds.

[0168]

[0169] The thiol group of iopamidol can also undergo a photocrosslinking reaction with the thiol group on gelatin to form a disulfide.

[0170]

[0171] The methyl allyl double bond on gelatin can undergo a photocrosslinking reaction with the double bond structure on iopamidol to form saturated alkanes.

[0172]

[0173] Click chemistry reactions with better biocompatibility are widely used. For example, the azide group introduced on gelatin can undergo an azide-alkynyl cycloaddition (CuAAC) reaction with the alkynyl group on iopamidol under copper catalysis to form a triazole structure. This reaction has the advantages of high yield, high regioselectivity and broad functional group tolerance.

[0174]

[0175] The excellent nucleophilicity of amino groups on gelatin can also be utilized. For example, the carboxyl groups on modified iopamidol can be activated by EDS / NHS to form activated esters, which then undergo nucleophilic substitution reactions with amino groups to form stable amide bonds.

[0176]

[0177] The aldehyde group on the modified iopamidol undergoes a nucleophilic addition-elimination reaction with the amino group on the gelatin to form an imine. This Schiff base can be further hydrogenated and reduced to form a secondary amine.

[0178]

[0179] The above reactions can all be applied to the covalent bonding between modified iopamidol and gelatin, thereby solving the problem of developer leakage in traditional physical mixing methods.

[0180] In this invention, the crosslinking includes at least one of ultraviolet light crosslinking and chemical crosslinking. Chemical crosslinking can avoid potential damage to the material from light exposure, and at the same time, by adjusting the amount of crosslinking agent, it enables precise control of the mechanical properties and degradation rate of the microspheres, adapting to the needs of different material characteristics.

[0181] In some specific embodiments of the present invention, the crosslinking is selected from ultraviolet light crosslinking. Specifically, modified iopamidol, gelatin (or modified gelatin) and photoinitiator are mixed in proportion, and a system with oil phase as continuous phase and raw material mixture as dispersed phase is constructed using a microfluidic chip. Microsphere particles are formed by ultraviolet light crosslinking.

[0182] Specifically, a microfluidic chip is used to interweave and shear the continuous phase and the dispersed phase in a microchannel. Under ultraviolet light irradiation, the dispersed phase droplets formed by shearing undergo a cross-linking reaction to obtain microsphere particles.

[0183] The continuous phase is selected from at least one of soybean oil and mineral oil;

[0184] The dispersed phase comprises an aqueous solution of modified iopamidol developer, biomaterial matrix, photoinitiator, and PBS buffer (pH 7.4);

[0185] In this invention, the dispersed phase comprises:

[0186] 1-10 wt% modified iopamidol developer;

[0187] 1-20 wt% biomaterial matrix;

[0188] 0.1-5 wt% photoinitiator;

[0189] The remaining amount of PBS buffer (pH 7.4) in aqueous solution.

[0190] In some specific embodiments of the present invention, the dispersed phase includes:

[0191] 5 wt% modified iopamidol developer;

[0192] 9wt% biomaterial matrix;

[0193] 0.3wt% photoinitiator;

[0194] The remaining amount of PBS buffer (pH 7.4) in aqueous solution.

[0195] The photoinitiator is selected from at least one of phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and 1-hydroxycyclohexylphenyl ketone.

[0196] The temperature of the crosslinking reaction is 40~90℃, and can be any value between 40, 55, 60, 70, 80, 90, or 40~90℃.

[0197] By controlling the microfluidic parameters mentioned above, grafting efficiency and product uniformity can be improved.

[0198] After obtaining the microspheres, post-processing is performed to obtain non-ionic microspheres that can be used for in vivo experiments.

[0199] In this invention, the post-processing includes centrifugation, washing, disinfection, and other post-processing to obtain purified and developed microspheres.

[0200] In large-scale production, microfluidic technology can be combined with membrane emulsification equipment to adjust parameters to ensure particle size uniformity (coefficient of variation ≤10%) and improve efficiency.

[0201] This invention uses modified iopamidol and modified or unmodified gelatin as the main raw materials. A one-pot process is employed to covalently graft modified iopamidol onto the gelatin polymer chain, imparting excellent imaging properties and hydrophilicity to the microspheres. Simultaneously, by adjusting the amount of modified iopamidol added, the density of the embolic microparticles and the X-ray imaging effect can be effectively controlled, ensuring clear monitoring of the microspheres' position and state in clinical applications. Furthermore, by adjusting the gelatin concentration, the degree of cross-linking of the modified gelatin microspheres can be controllably adjusted. Different degrees of cross-linking directly affect the mechanical properties and degradation rate of the microspheres, allowing them to adapt to different treatment needs.

[0202] This invention presents a technical solution for introducing the clinically safe, non-ionic, hydrophilic contrast agent iopamidol into a gelatin-based biodegradable microsphere matrix via chemical bonding. The invention utilizes the inherent hydrophilic serine amino alcohol groups of iopamidol and the hydrophilic side chains introduced through modification of iopamidol to improve the hydrophobicity of the microspheres. Through a covalent bonding process, the distribution density of the contrast agents and the mechanical properties of the microspheres are precisely controlled. While retaining excellent X-ray imaging performance, this solution addresses the aggregation tendency and biocompatibility issues of traditional contrast-enhancing microspheres, enabling real-time visual monitoring of the embolization process. The contrast-enhancing embolization microsphere system of this invention combines biodegradability, stable imaging, and good biosafety. It is not only suitable for permanent embolization treatment but can also be extended to temporary embolization, tissue engineering, and other fields, providing a novel solid contrast-enhancing embolic agent with both precise imaging and safe degradation properties for minimally invasive interventional treatment of diseases such as tumor intervention and vascular malformations.

[0203] To further understand the present invention, the following embodiments illustrate the developer and its preparation method, the nonionic developer microspheres and their preparation method, and their applications. The scope of protection of the present invention is not limited by the following embodiments.

[0204] Example 1:

[0205] Step 1: Preparation of modified iopamidol IV-1

[0206] The chemical equation for the preparation of modified iopamidol IV-1 is as follows.

[0207]

[0208] The specific preparation method includes the following steps: using TMS-protected iopamidol I-1 (10.0 g) and carboxylic acid II-1 (1.9 g) as reactants, N,N'-dicyclohexylcarbodiimide (DCC) (2.3 g) as the activating agent of the carboxylic acid, 4-dimethylaminopyridine (DMAP) (114.7 mg) as the catalyst for the esterification reaction, and dried tetrahydrofuran (THF) (50 mL) as the reaction solvent, the reaction was carried out at room temperature for 12 hours under an inert gas atmosphere. After the reaction, the white precipitate dicyclohexylurea (DCU) was removed by filtration, and the filter cake was washed with a small amount of dichloromethane; the filtrate was washed successively with 10% citric acid aqueous solution (to wash away DMAP), saturated sodium bicarbonate aqueous solution (to wash away residual carboxylic acid), and saturated brine (to wash away water-soluble impurities), dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product III-1 was separated by silica gel column chromatography to obtain TMS-protected oily modified iopamidol III-1 (9.0 g, 78% yield). 1 H-NMR, 13 C-NMR and HRMS characterization confirmed the structure was consistent with the indicated structure. Next, in a dry 100 mL reaction flask, TMS-protected modified iopamidol III-1 (9.0 g) was added, dissolved in 50 mL of anhydrous THF, and the mixture was heated under an argon atmosphere. o Under C conditions, 8.8 mL of TBAF (1 M in THF) solution was slowly added dropwise. After slowly restoring to room temperature and stirring for 1 hour, excess TBAF was quenched by adding 30 mL of saturated ammonium chloride aqueous solution. The mixture was extracted with ethyl acetate (3 × 40 mL), and the organic phases were combined. After drying over anhydrous sodium sulfate, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain the final modified iopamidol IV-1 (5.9 g, 86% yield). The overall yield of the two-step reaction was 67%. The 1H and 1C NMR spectra and high-resolution data of the obtained modified iopamidol IV-1 are as follows: 1H NMR (600 MHz, Methanol-d4) δ 6.33 (dd, J = 17.4, 1.4 Hz, 1H), 6.11 (dd,J = 17.4, 10.4 Hz, 1H), 5.84 (dd, J = 10.4, 1.4 Hz, 1H), 5.33 – 5.26 (m, 1H),4.12 (t, J = 6.6 Hz, 2H), 4.05 – 4.01 (m, 2H), 3.85 – 3.77 (m, 4H), 3.74 –3.68 (m, 4H), 2.56 – 2.41 (m, 2H), 1.72 – 1.63 (m, 4H), 1.62 – 1.57 (m, 3H),1.47 – 1.38 (m, 2H) ppm; 13 C NMR (150 MHz, Methanol-d4) δ 172.7, 170.6, 170.3,166.4, 149.8, 142.6, 130.3, 128.3, 98.3, 97.7, 88.9, 69.9(2), 69.8(8), 64.2,61.9, 61.8, 44.2, 44.1, 33.4(2), 33.3(5), 28.1(1), 28.0(8), 25.2(1), 25.1(9),24.9, 24.8, 24.2, 23.9, 23.7, 22.0, 21.8, 20.9, 20.7, 17.1(2), 17.0(8) ppm;HRMS (m / z) calculated for C 26 H 34 I3N3O 11 Na + 967.9220, found 967.9214 [M+Na] + .

[0209] Step 2: Preparation of developing microspheres

[0210] After obtaining the modified iopamidol IV-1, the next step is to prepare the developing microspheres.

[0211] First, the synthesis of methacrylamide gelatin (GelMA) was carried out. 5 g of gelatin was weighed and added to 50 mL of deionized water. The solution swelled at room temperature for 1 h, then transferred to a 50 °C constant temperature water bath and stirred continuously until completely dissolved. Methacrylamide anhydride was added dropwise to the gelatin solution at a rate of 0.2 mL / min, with a cumulative addition of 2 mL. The mixture was then placed in a 50 °C constant temperature shaker and reacted for 4 h. After the reaction, 200 mL of 50 °C deionized water was added to dilute the reaction solution. The diluted solution was transferred to a dialysis bag with a molecular weight cutoff of 8–14 kDa and dialyzed against 50 °C deionized water for 4 days, with the deionized water changed every 4 h. After dialysis, the solution in the bag was centrifuged at 4500 rpm for 10 min. The supernatant was pre-frozen overnight at -80 °C and then freeze-dried to obtain a solid powder, which was the methacrylamide gelatin (GelMA).

[0212] Subsequently, developing microspheres were prepared using a PDMS-based microfluidic chip. Soybean oil was used as the continuous phase (flow rate 20 μL / min), and a PBS buffer solution containing GelMA (9.0 wt%), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (0.3 wt%), and modified iopamidol IV-1 (5.0 wt%) was used as the dispersion phase (flow rate 6 μL / min). The microspheres were obtained by passing droplets through a 405 nm UV-crosslinked serpentine channel at 55°C. The obtained developing microspheres had a diameter of approximately 620 μm (minor axis diameter) and a length of approximately 1000 μm. The density was 1.241 g / mL. Images of the obtained nonionic developing microspheres observed under an optical microscope are shown below. Figure 1 As shown. Figure 1 This is an optical microscope image of the non-ionic microspheres prepared in Example 1. Figure 2 The image shows an X-ray image of the non-ionic microspheres prepared in Example 1. Figure 3 The image shows a micro-computed tomography (CT) image of the non-ionic radiopaque microspheres prepared in Example 1.

[0213] Example 2:

[0214] Step 1: Preparation of modified iopamidol IV-2

[0215] The chemical equation for the preparation of modified iopamidol IV-2 is as follows:

[0216]

[0217] The preparation process of modified iopamidol IV-2 is represented by the following chemical equation: Figure 6As shown, the specific preparation method includes the following steps: using phenylboronic acid ester-protected iopamidol I-2 (10.0 g) and carboxylic acid II-2 (2.1 g) as reactants, N,N'-diisopropylcarbodiimide (DIC) (1.6 g) as the activating agent of the carboxylic acid, 4-dimethylaminopyridine (DMAP) (128.1 mg) as the catalyst for the esterification reaction, and dried tetrahydrofuran (THF) (50 mL) as the reaction solvent, the reaction was carried out at room temperature under an inert gas atmosphere for 12 hours. After the reaction was completed, saturated ammonium chloride solution (50 mL) was added to quench the reaction, and the aqueous phase containing a large amount of diisopropylurea was removed by separation. The organic phase was washed successively with 10% citric acid solution, saturated sodium bicarbonate solution, and saturated brine. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum evaporation, and the oily modified iopamidol III-2 (7.6 g, 65% yield) protected by phenylboronic acid ester was obtained by silica gel column chromatography. Next, in a 100 mL reaction flask, 7.6 g of modified iopamidol III-2 protected by phenylboronic acid ester was added, dissolved in 40 mL of DMF. Hydrochloric acid aqueous solution (1.0 M, 14 mL) was slowly added at room temperature. TLC monitoring continued until the starting material was completely hydrolyzed. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 50 mL), and the organic phases were combined. The organic phase was then extracted again with water (3 × 30 mL) to remove residual DMF solvent. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain the final modified iopamidol IV-2 (5.6 g, 87% yield). The overall yield of the two-step reaction was 57%. The 1H and 1C NMR spectra and high-resolution data of the obtained compound IV-2 are as follows: 1 H NMR (600 MHz, Methanol-d4) δ 6.17 – 5.99 (m, 2H), 5.59 – 5.47 (m, 1H), 5.26 – 5.18 (m, 1H), 4.02 – 3.90 (m, 2H), 3.79 – 3.69 (m, 4H), 3.69 – 3.60 (m, 4H), 3.18 – 3.09 (m, 2H), 2.49 – 2.31 (m, 2H), 1.66 – 1.38 (m, 7H), 1.36 – 1.25 (m, 2H) ppm; 13C NMR (150 MHz, Methanol-d4) δ 172.5, 170.4, 170.1, 166.4, 149.7, 142.0,130.3(4), 130.3(2), 125.0(0), 124.9(8), 97.2, 88.6, 69.5, 59.4, 52.5, 38.5,33.0, 28.3, 25.7, 23.8, 16.6 ppm; HRMS (m / z) calculated for C 26 H 35 I3N4O 10 Na + 966.9379, found 966.9386 [M+Na] + .

[0218] Step 2: Preparation of developing microspheres

[0219] After obtaining the modified iopamidol IV-2, the next step is to prepare the developing microspheres.

[0220] First, the synthesis of methacrylamide gelatin (GelMA) was carried out. 5 g of gelatin was weighed and added to 50 mL of deionized water. The solution swelled at room temperature for 1 h, then transferred to a 50 °C constant temperature water bath and stirred continuously until completely dissolved. Methacrylamide anhydride was added dropwise to the gelatin solution at a rate of 0.2 mL / min, with a cumulative addition of 2 mL. The mixture was then placed in a 50 °C constant temperature shaker and reacted for 4 h. After the reaction, 200 mL of 50 °C deionized water was added to dilute the reaction solution. The diluted solution was transferred to a dialysis bag with a molecular weight cutoff of 8–14 kDa and dialyzed against 50 °C deionized water for 4 days, with the deionized water changed every 4 h. After dialysis, the solution in the bag was centrifuged at 4500 rpm for 10 min. The supernatant was pre-frozen overnight at -80 °C and then freeze-dried to obtain a solid powder, which was the methacrylamide gelatin (GelMA).

[0221] Subsequently, developing microspheres were prepared using a PDMS-based microfluidic chip. Soybean oil was used as the continuous phase (flow rate 25 μL / min), and a PBS buffer solution containing GelMA (9.0 wt%), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (0.3 wt%), and modified iopamidol IV-2 (5.0 wt%) was used as the dispersed phase (flow rate 6 μL / min). The microspheres were obtained by passing droplets through a 405 nm UV-crosslinked serpentine channel at 55°C. The obtained developing microspheres had a diameter of approximately 600 μm (minor axis diameter) and a length of approximately 1100 μm. The density was 1.241 g / mL. Images of the obtained nonionic developing microspheres observed under an optical microscope are shown below. Figure 4 As shown. Figure 4 This is an optical microscope image of the non-ionic microspheres prepared in Example 2. Figure 5 The image shows an X-ray image of the non-ionic microspheres prepared in Example 2. Figure 6 The image shows a micro-computed tomography (CT) image of the non-ionic imaging microspheres prepared in Example 2.

[0222] Example 3:

[0223] Step 1: Preparation of modified iopamidol IV-3

[0224] The chemical equation for the preparation of modified iopamidol IV-3 is as follows:

[0225]

[0226] The specific preparation method includes the following steps: using phenylboronic acid ester-protected iopamidol I-2 (10.0 g) and carboxylic acid II-3 (3.2 g) as starting materials, N,N'-diisopropylcarbodiimide (DIC) (1.6 g) as the activating agent of the carboxylic acid, 4-dimethylaminopyridine (DMAP) (128.1 mg) as the catalyst for the esterification reaction, and dried tetrahydrofuran (THF) (50 mL) as the reaction solvent, the reaction was carried out at room temperature under an inert gas atmosphere for 12 hours. After the reaction was completed, saturated ammonium chloride solution (50 mL) was added to quench the reaction, and the aqueous phase containing a large amount of diisopropylurea was removed by separation. The organic phase was washed successively with 10% citric acid solution, saturated sodium bicarbonate solution, and saturated brine. After drying with anhydrous sodium sulfate, the solvent was removed by vacuum distillation, and silica gel column chromatography was used to separate the phenylboronic acid ester-protected oily modified iopamidol III-3 (9.4 g, 74% yield). Product III-3 was subjected to... 1 H-NMR, 13C-NMR and HRMS characterization confirmed the structure. Next, in a 100 mL reaction flask, 8.7 g of phenylboronic acid-protected modified iopamidol III-3 was added, dissolved in 40 mL of DMF. Hydrochloric acid aqueous solution (1.0 M, 16 mL) was slowly added at room temperature. TLC was performed until the starting material was completely hydrolyzed. After the reaction, the mixture was extracted with ethyl acetate (3 × 50 mL), and the organic phases were combined. The organic phase was then extracted again with water (3 × 40 mL) to remove residual DMF solvent. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain the final modified iopamidol IV-3 (6.0 g, 75% yield). The overall yield of the two-step reaction was 56%. The 1H and 1C NMR spectra and high-resolution data of the obtained compound IV-3 are as follows: 1 H NMR (600 MHz, Methanol-d4)δ 6.42 (dd, J = 17.4, 1.4 Hz, 1H), 6.20 (dd, J = 17.4, 10.4 Hz, 1H), 5.93(dd, J = 10.4, 1.4 Hz, 1H), 5.44 – 5.37 (m, 1H), 4.34 – 4.29 (m, 2H), 4.14 –4.06 (m, 2H), 3.93 – 3.72 (m, 12H), 3.69 – 3.58 (m, 8H), 2.87 – 2.69 (m, 2H),1.69 – 1.64 (m, 3H) ppm; 13 C NMR (150 MHz, Methanol-d4) δ 170.8, 170.5, 170.3,166.3, 150.2, 142.3, 130.5, 128.1, 97.5, 88.9, 70.2(1), 70.1(8), 70.1(6),70.1, 68.7, 66.2, 63.5, 59.8, 59.5, 53.2, 53.1, 52.9, 34.6, 34.5, 16.9 ppm;HRMS (m / z) calculated for C 29 H 40 I3N3O 14 Na + 1057.9537, found 1057.9529 [M+Na] + .

[0227] Step 2: Preparation of developing microspheres

[0228] After obtaining modified iopamidol IV-3 and azide-modified gelatin, the next step is to prepare developing microspheres.

[0229] First, the synthesis of methacrylamide gelatin (GelMA) was carried out. 5 g of gelatin was weighed and added to 50 mL of deionized water, allowing it to swell at room temperature for 1 h. Then, it was transferred to a 50 ℃ constant temperature water bath and stirred continuously until completely dissolved. Methacrylamide anhydride was added dropwise to the gelatin solution at a rate of 0.2 mL / min, with a cumulative addition of 2 mL. The mixture was then placed in a 50 ℃ constant temperature shaker and reacted for 4 h. After the reaction, 200 mL of 50 ℃ deionized water was added to dilute the reaction solution. The diluted solution was transferred to a dialysis bag with a molecular weight cutoff of 8–14 kDa and dialyzed in 50 ℃ deionized water for 4 days, changing the deionized water every 4 h. After dialysis, the solution in the bag was centrifuged at 4500 rpm for 10 min, and the supernatant was pre-frozen overnight at -80 ℃. After freeze-drying, a solid powder was obtained, which was the methacrylamide gelatin (GelMA).

[0230] Subsequently, developing microspheres were prepared using a PDMS-based microfluidic chip. Soybean oil was used as the continuous phase (flow rate 25 μL / min), and a PBS buffer solution containing GelMA (9.0 wt%), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (0.3 wt%), and modified iopamidol IV-3 (5.0 wt%) was used as the dispersion phase (flow rate 6 μL / min). The microspheres were obtained by passing droplets through a 405 nm UV-crosslinked serpentine channel at 55°C. The obtained developing microspheres had a diameter of approximately 600 μm (minor axis diameter) and a length of approximately 1000 μm. The density was 1.22 g / mL. Images of the obtained nonionic developing microspheres observed under an optical microscope are shown below. Figure 7 As shown. Figure 7 This is an optical microscope image of the non-ionic microspheres prepared in Example 3. Figure 8 This is an X-ray imaging image of the non-ionic microspheres prepared in Example 3. Figure 9 The image shows a micro-computed tomography (CT) image of the non-ionic imaging microspheres prepared in Example 3. Figure 10 The non-ionic radiopaque microspheres prepared in Example 3 were injected into the ears of New Zealand rabbits, and the degradation and radiopaque changes of the radiopaque microspheres were detected by micro-computed tomography (CT) at 0, 7, 14, 30, 60 and 90 days.

[0231] Depend on Figure 10It is known that the changes in the imaging performance of the biodegradable imaging microspheres provided by this invention in living animals (such as rabbit ears) are linearly related to their degree of degradation. Therefore, the imaging changes shown by computed tomography (CT) scans can reasonably infer the degree of degradation of the microspheres in blood vessels, enabling non-invasive and real-time assessment of the absorption of biodegradable materials in vivo.

[0232] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A developer, characterized in that, The developer is modified iopamidol, and the modified iopamidol has the structure shown in Formula IV: Formula IV In equation IV, R 2 Selected from at least one group having the following structure: n is any integer greater than or equal to 1; R 3 Selected from at least one group having the following structure: 。 2. The developer according to claim 1, characterized in that, The developer is selected from at least one compound having the following structure: 。 3. A method for preparing a developer as described in claim 1 or 2, characterized in that, Includes the following steps: Iopamidol with protected hydroxyl groups was condensed with a carboxylic acid compound and then deprotected to obtain a developer. The filamentous amino alcohol hydroxyl group protected by iopamidol has the structure shown in Formula I: Equation I The R 1 Independently selected from groups having the following structures: Or, R 1 The oxygen atoms connected to them form a ring, and the ring has any of the following structures: ； The carboxylic acid compound has the structure shown in Formula II: Formula II In Equation II, R 2 Selected from at least one group having the following structure: n is any integer greater than or equal to 1; R 3 Selected from at least one group having the following structure: 。 4. The preparation method according to claim 3, characterized in that, The condensation reaction is carried out under conditions of catalyst and activator, wherein some condensation conditions require the addition of a base to replace the catalyst; The catalyst is selected from 4-dimethylaminopyridine; The base is selected from at least one of N,N-diisopropylethylamine, triethylamine, N-methyldiisopropylamine, pyridine, and N-methylmorpholine; The activator is selected from carboxyl activators, and the carboxyl activator is selected from at least one of the following: dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, cyclohexylisobutylcarbodiimide, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate. The deprotection reagent used is selected from tetrabutylammonium fluoride, hydrochloric acid, and sodium hydroxide.

5. A non-ionic imaging microsphere, characterized in that, It is prepared from a developer and a biomaterial matrix, wherein the developer is selected from the developer described in claim 1 or 2.

6. The non-ionic imaging microspheres according to claim 5, characterized in that, The biomaterial matrix is ​​selected from one or more of natural biodegradable polymers and / or synthetic polymers; The natural biodegradable polymer is selected from gelatin, chitosan, sodium hyaluronate, sodium alginate, and carrageenan; The synthetic polymer is selected from polylactic acid-hydroxyacetic acid copolymer, polycaprolactone, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, and polyacrylamide.

7. The non-ionic imaging microspheres according to claim 6, characterized in that, The gelatin is selected from at least one of unmodified gelatin and modified gelatin; The modification is as follows: Modify the -NH2 group in the gelatin structure with any of the following groups: 、 、 ； n is any integer greater than or equal to 1; And / or, modify the -COOH group in the gelatin structure with the following groups: 。 8. The non-ionic imaging microspheres according to claim 5, characterized in that, In the nonionic developing microspheres, the loading of the developer is 5-20 wt%; The biomaterial matrix is ​​selected from gelatin, and the degree of cross-linking of the gelatin is 30%-70%.

9. The non-ionic imaging microspheres according to claim 5, characterized in that, The non-ionic developing microspheres have an ellipsoidal structure, with a minor axis diameter of 100-700 μm, a length of 500-1200 μm, and a density of 1.05-1.3 g / cm³. 3 .

10. The non-ionic imaging microspheres according to claim 5, characterized in that, The non-ionic radiopaque microspheres also include a drug, which is loaded in the biomaterial matrix.

11. A method for preparing nonionic imaging microspheres as described in any one of claims 5 to 10, characterized in that, Includes the following steps: A) The developer is cross-linked with the biomaterial matrix using a microfluidic method to obtain microspheres, which are non-ionic developer microspheres; B) The microspheres are post-processed to obtain non-ionic radiopaque microspheres that can be used for in vivo experiments.

12. The preparation method according to claim 11, characterized in that, The crosslinking includes at least one of ultraviolet light crosslinking and chemical crosslinking.

13. The preparation method according to claim 11, characterized in that, Step A) includes: Using a microfluidic chip, the continuous phase and the dispersed phase are intersected and sheared in a microchannel. Under ultraviolet light irradiation, the dispersed phase droplets formed by shearing undergo a cross-linking reaction to obtain microsphere particles. The continuous phase is selected from at least one of soybean oil and mineral oil; The dispersed phase comprises an aqueous solution of modified iopamidol developer, biomaterial matrix, photoinitiator, and PBS buffer (pH 7.4); The photoinitiator includes, but is not limited to, at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and 1-hydroxycyclohexylphenyl ketone; The flow rate of the continuous phase is 20-30 μL / min, and the flow rate of the dispersed phase is 2-12 μL / min; The cross-linking reaction is carried out at a temperature of 40~90℃.

14. The use of the non-ionic radiopaque microspheres as described in any one of claims 5 to 10 in the preparation of radiotherapy, chemotherapy and embolization combined therapy drugs and radiopaque diagnostic reagents for tumors.

Images