Ultrasonic-triggered enzymatic cross-linked hydrogel embolization agent and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses an ultrasonic-triggered enzymatic cross-linked hydrogel embolization agent and a preparation method thereof. The invention provides an ultrasonic-triggered enzymatic cross-linked hydrogel embolization agent. The ultrasonic-triggered enzymatic cross-linked hydrogel embolization agent comprises oxidized macromolecules and derivatives thereof, nitrogen-containing macromolecules and derivatives thereof, ultrasonic-responsive lipidosome and a solvent, the ultrasonic responsive liposome is a spherical liposome and is filled with a secondary cross-linking agent and phase change liquid drops. According to the application, a'step-by-step cross-linking 'strategy is adopted, the oxidized polymer and the derivative thereof are subjected to primary cross-linking with the nitrogen-containing polymer and the derivative thereof, and then the ultrasonic responsive liposome is triggered by ultrasonic to release the internally filled secondary cross-linking agent, so that secondary cross-linking is completed; finally, the hydrogel embolism agent with strong injectability, high stability after secondary crosslinking and a developing function is prepared, and the technical problems that an existing hydrogel embolism agent is weak in stability, reversible in gel form, incapable of achieving on-demand reinforcement and incapable of developing are solved.

Description

Technical Field

[0001] This application relates to biomedical materials, and more particularly to an ultrasound-triggered enzymatic cross-linked hydrogel embolizer and its preparation method. Background Technology

[0002] Transcatheter embolization (TAE), as a core technology of minimally invasive interventional therapy, plays an irreplaceable role in the treatment of diseases such as tumors, arteriovenous malformations, and aneurysms. The efficacy of TAE largely depends on the performance of the embolic material. An ideal embolic material not only needs to have good injectability, that is, be able to be smoothly delivered through a microcatheter, and adapt to different vessel shapes and sizes in complex vascular anatomy, but also need to rapidly transform into a strong solid in the target vessel to withstand continuous blood flow pressure and achieve long-term stable vascular occlusion.

[0003] Injectable hydrogels, as novel embolic agents, are gradually becoming a hot research topic. Due to their excellent biocompatibility, biodegradability, and tunable physicochemical properties, hydrogels are ideal candidates for embolic agents. Hydrogels can slowly degrade in vivo through chemical or physical cross-linking, thereby reducing the risk of long-term embolic agent residue, and their physical properties such as hardness and viscosity can be adjusted according to needs. However, the application of injectable hydrogels in embolization therapy still faces some technical obstacles: physically cross-linked hydrogels (such as thermosensitive hydrogels) generally have weak mechanical strength and are easily eroded by blood flow, losing stability; while chemically cross-linked hydrogels (such as hydrogels based on a single Schiff base reaction) have the problem of dynamic reversibility of the cross-linked network, easily undergoing hydrolytic relaxation under long-term physiological conditions, leading to a decline in mechanical properties after embolization, and even the possibility of vascular recanalization. More importantly, existing hydrogel embolization systems are mostly autonomous and uncontrollable, lacking the intelligent response capability to achieve "on-demand reinforcement" in complex blood flow environments; in addition, embolization agents usually lack endogenous imaging capabilities, making it difficult to monitor and evaluate the embolization range and effect in real time during treatment, affecting the accuracy of treatment.

[0004] The aforementioned limitations mean that injectable hydrogel embolic agents have not yet fully realized their potential in clinical applications, and there is an urgent need to develop more ideal embolic agents. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide an ultrasound-triggered enzymatic crosslinking hydrogel embolization agent and its preparation method, in order to solve the technical problems of existing hydrogel embolization agents, such as weak stability, reversible gel morphology, inability to achieve on-demand reinforcement, and inability to be visualized.

[0006] To achieve the above technical objectives, this application provides an ultrasound-triggered enzymatic cross-linking hydrogel embolizer, comprising oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives, ultrasound-responsive liposomes, and a solvent;

[0007] Oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives undergo a first cross-linking and solidification process via Schiff base reaction to form a hydrogel matrix.

[0008] The ultrasound-responsive liposomes are spherical liposomes, and the interior of the spherical liposomes is filled with a secondary cross-linking agent and a phase change droplet;

[0009] In this process, the phase change droplets are used to receive ultrasonic signals and change phase to gaseous state, thereby destroying the structure of the spherical liposomes and releasing the secondary crosslinking agent; the secondary crosslinking agent is used to react with the residual active groups in the hydrogel matrix to achieve the second crosslinking and curing of the hydrogel matrix.

[0010] Furthermore, in the enzyme-crosslinked hydrogel embolization agent, the volume fraction of oxidized polymers and their derivatives is 1-2%, the volume fraction of nitrogen-containing polymers and their derivatives is 5-10%, and the volume fraction of ultrasound-responsive liposomes is 1-2%.

[0011] Furthermore, the phase change droplets include at least one of perfluorohexane and perfluoropentane; and / or, the oxidized polymers and their derivatives are prepared by oxidation of natural polymers, and the natural polymers include at least one of hyaluronic acid, sodium alginate, chondroitin sulfate, carboxymethyl cellulose, and dextran; and / or, the nitrogen-containing polymers and their derivatives include at least one of amination polymers and their derivatives, collagen, ε-polylysine, and chitosan; and / or, the secondary cross-linking agent includes at least one of transglutaminase, thrombin, and horseradish peroxidase.

[0012] Furthermore, the ultrasound-responsive liposomes are prepared from phospholipids with a phase transition temperature; and / or, the ultrasound-responsive liposomes include an additive that lowers the cavitation threshold; and / or, the ultrasound-responsive liposomes are loaded with a sonicator; and / or, the surface of the ultrasound-responsive liposomes is coupled with ultrasound microbubbles, which are encapsulated with a biocompatible gas; and / or, the interior of the ultrasound-responsive liposomes is filled with a biocompatible gas.

[0013] Furthermore, the phospholipids having a phase transition temperature include at least one of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and palmitoyloleoylphosphatidylcholine; and / or, the additives for reducing the cavitation threshold include at least one of cholesterol and surfactants; and / or, the sonication sensitizers include at least one of protoporphyrin and rose red; and / or, the biocompatible gases include at least one of air, oxygen, and carbon dioxide.

[0014] Furthermore, it also includes nonionic contrast agents and / or particulate contrast agents; nonionic contrast agents include at least one of iopamidol, iodixanol, iopromide, and iohexol; particulate contrast agents include at least one of barium sulfate micron particles, tantalum nanoparticles, and liquid metal.

[0015] Furthermore, the preparation method of oxidized polymers and their derivatives is as follows: oxidized polymers and their derivatives are dissolved in water, an oxidant is added and reacted in the dark, and after the reaction is terminated, they are dialyzed and freeze-dried to obtain oxidized polymers and their derivatives.

[0016] Furthermore, the preparation method of nitrogen-containing polymers and their derivatives is as follows: dissolve the polymers and their derivatives in buffer solution, add amination reagent and activating solution to react, adjust the pH to a preset range, and after reaction, dialyze and freeze dry to obtain amination polymers and their derivatives.

[0017] Furthermore, the preparation method of ultrasound-responsive liposomes is as follows: phospholipids and their derivatives are dissolved in an organic solvent, and a lipid film is obtained by rotary evaporation under reduced pressure. A suspension is obtained by elution with a secondary crosslinking agent solution. Phase change droplets are added to the suspension and mixed evenly. After centrifugation, washing, and resuspension, ultrasound-responsive liposomes loaded with secondary crosslinking agent and phase change droplets are obtained.

[0018] This application provides a method for preparing an ultrasound-triggered enzymatic cross-linked hydrogel embolizer, comprising the following steps: mixing oxidized polymers and their derivatives, and nitrogen-containing polymers and their derivatives with solvents respectively to obtain solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives; mixing the solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives with ultrasound-responsive liposomes to obtain an ultrasound-triggered enzymatic cross-linked hydrogel embolizer.

[0019] In summary, this application provides an ultrasound-triggered enzymatic crosslinking hydrogel embolizing agent, comprising an oxidized polymer and its derivatives, a nitrogen-containing polymer and its derivatives, an ultrasound-responsive liposome, and a solvent; the oxidized polymer and its derivatives and the nitrogen-containing polymer and its derivatives undergo a first crosslinking and curing reaction via a Schiff base reaction to form a hydrogel matrix; the ultrasound-responsive liposome is a spherical liposome, the interior of which is filled with a secondary crosslinking agent and a phase change droplet; wherein, the phase change droplet is used to receive the ultrasound signal and change phase to a gaseous state to destroy the structure of the spherical liposome and release the secondary crosslinking agent; the secondary crosslinking agent is used to react with the residual active groups in the hydrogel matrix to achieve a second crosslinking and curing of the hydrogel matrix.

[0020] The ultrasound-triggered enzymatic cross-linked hydrogel embolization agent provided in this application has the following two advantages: First, by utilizing the rapid and mild nature of the Schiff base reaction, the embolization agent can be rapidly injected and initially formed, ensuring good injectability and effectively solving the problem of reflux prevention during injection, thus ensuring the convenience and safety of clinical operation. Second, by innovatively using external ultrasound triggering, a stable and irreversible covalent cross-linked network catalyzed by transglutaminase is constructed in situ within the initially formed gel network. This process significantly improves the mechanical strength and anti-degradation ability of the embolization agent, thereby enhancing its long-term stability and effectively avoiding the clinical risk of vascular recanalization. Secondly, this application endows the embolization treatment process with the ability of "precise time and temperature control" and the advantages of "integrated diagnosis and treatment": On the one hand, the phase change droplets inside the ultrasound-responsive liposome undergo a bubble reaction under the action of ultrasound, which can directly achieve the imaging effect, making it convenient for clinical real-time monitoring of the embolization location and effect; on the other hand, compared with the autonomous and uncontrollable gelation process in the prior art, the secondary cross-linking reaction of this application is triggered by ultrasound, which can precisely control the timing and location of the cross-linking reaction, and can reinforce the key embolization sites of blood vessels at specific points, greatly improving the adaptability of the embolizing agent to the complex blood flow environment, and further ensuring the clinical treatment effect.

[0021] Compared with existing technologies, the embolization agent provided in this application has the advantages of injectability, strong mechanical properties after secondary cross-linking, and imaging effect. It is clearly visible under dual-modal imaging of X-ray and ultrasound, providing a reliable guarantee for the visual monitoring of the embolization treatment process. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A diagram illustrating the preparation and curing mechanism of the hydrogel embolizing agent provided in Example 1;

[0024] Figure 2 The figures show the morphology and related performance characteristics of LTP and Lip; among them, Figure 2 In the diagram, A represents a comparison of the particle size distribution of the two types of liposomes. Figure 2 B in the diagram represents the potential comparison. Figure 2 C in the figure represents a comparison of the appearance stability of the two liposomes after one week. Figure 2 D in the figure represents a comparison of the intensity distribution of the two types of liposomes after one week. Figure 2 E in the figure represents a comparison of the potential changes of the two types of liposomes after one week.

[0025] Figure 3 This is a comparison of the in vitro ultrasound contrast effects of LTP and the commercial ultrasound contrast agent SonoVe.

[0026] Figure 4 The figures show a comparison of the characterization of modified hydrogel matrices; among them, Figure 4 In the figure, A represents the characterization of the curing properties of hydrogels prepared by gel and HA at simulated human physiological temperature; Figure 4 B-1 in the figure is a comparison of the FTIR spectra of Gel and AG; Figure 4 C-1 in the figure represents the ¹H-NMR spectrum of Gel and AG; Figure 4 B-2 in the figure is a comparison of the FTIR spectra of OHA and HA; Figure 4 C-2 in the figure represents the ¹H-NMR spectra of OHA and HA; Figure 4 D in the diagram represents the injectability properties of the precursor hydrogel. Figure 4 E in the figure represents the relationship between the fluidity of the hydrogel and the shear rate. Figure 4 In this context, F represents the result of a strain cyclic experiment; Figure 4 G in the figure is a schematic diagram of the change in storage modulus of the precursor hydrogel under ultrasonic stimulation.

[0027] Figure 5 This image shows an injection force testing device and imaging effect characterization diagram for hydrogel embolic agents; among which, Figure 5 In the diagram, A represents the injection force testing device. Figure 5 In the image, B represents the X-ray and ultrasound imaging effects of hydrogel embolization agents with different concentrations of iohexol. Figure 5 C in the figure is a comparison of the ultrasonic imaging effects of the hydrogel embolization agent and SonoVe.

[0028] Figure 6 The graphs represent the biocompatibility and coagulation capacity of the hydrogel embolization agent; among them, Figure 6 Figure A in the graph shows the cell viability detection results under different concentrations of hydrogel embolizing agent extract. Figure 6 In the image, B represents the staining effect of live and dead cells. Figure 6 C in the diagram represents the hemolysis rate of the hydrogel embolization agent; Figure 6 D in the figure represents a comparison of the coagulation ability of hydrogel embolic agents.

[0029] Figure 7 Images and related characterizations of hydrogel embolic agents in a rabbit kidney embolism model; among them... Figure 7 In the image, A represents the ultrasound-triggered liposome rupture and imaging effect. Figure 7 B in the diagram represents the location of the microcatheter and the injection path of the hydrogel embolization agent under X-ray fluoroscopy. Figure 7 C in the image represents a comparison of DSA angiography and Doppler ultrasound blood flow signals before and after embolization. Figure 7 D in the image represents a CT scan of vital organs 28 days post-surgery. Detailed Implementation

[0030] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments in this application specification, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection claimed in this application.

[0031] In the description of this application, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," indicating orientation or positional relationships, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this application. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0032] Unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections or electrical connections; they can refer to direct connections or indirect connections through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0033] The raw materials used in this invention are not particularly restricted in their source; they can be purchased on the market or prepared using conventional methods known to those skilled in the art.

[0034] This application provides an ultrasound-triggered enzymatic cross-linking hydrogel embolizer, comprising oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives, ultrasound-responsive liposomes, and a solvent;

[0035] Oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives undergo a first cross-linking and solidification process via Schiff base reaction to form a hydrogel matrix.

[0036] The ultrasound-responsive liposomes are spherical liposomes, and the interior of the spherical liposomes is filled with a secondary cross-linking agent and a phase change droplet;

[0037] In this process, the phase change droplets are used to receive ultrasonic signals and change phase to gaseous state, thereby destroying the structure of the spherical liposomes and releasing the secondary crosslinking agent; the secondary crosslinking agent is used to react with the residual active groups in the hydrogel matrix to achieve the second crosslinking and curing of the hydrogel matrix.

[0038] It should be noted that this application utilizes oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives to form injectable hydrogels, and encapsulates secondary cross-linking agents and phase change droplets via liposome carriers to achieve precise control of the embolization reaction. This embolic agent undergoes secondary cross-linking within the target blood vessel via ultrasound stimulation, rapidly consolidating and stabilizing the embolization effect, thereby improving treatment precision and safety, and providing an innovative solution for interventional treatment of diseases such as tumors and aneurysms. Specifically, oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives form dynamic imine bonds (-C=N-) through a Schiff base reaction, enabling rapid transformation from solution to gel, giving them excellent injectability and rapid shaping capabilities. The liposome carrier encapsulates phase change droplets and secondary cross-linking agents, which release the secondary cross-linking agent under ultrasound stimulation, triggering the secondary cross-linking reaction. Furthermore, the phase change droplets, after phase change, also possess ultrasound imaging capabilities, providing potential endogenous imaging functions. The ultrasound-triggered secondary cross-linking reaction mechanism effectively enhances the mechanical strength of the embolic agent, enabling it to precisely achieve the embolization effect within the target blood vessel. The principle diagram can be found in [reference needed]. Figure 1 .

[0039] In some embodiments, the volume fraction of oxidized polymers and their derivatives in the enzymatically cross-linked hydrogel embolizing agent is 1-2%, the volume fraction of nitrogen-containing polymers and their derivatives is 5-10%, and the volume fraction of ultrasound-responsive liposomes is 1-2%.

[0040] In some preferred embodiments, the volume fraction of oxidized polymers and their derivatives in the enzymatically crosslinked hydrogel embolization agent is 1.5%, the volume fraction of nitrogen-containing polymers and their derivatives is 7%, and the volume fraction of ultrasound-responsive liposomes is 1.17%.

[0041] In some embodiments, the phase change droplets include at least one of perfluorohexane and perfluoropentane;

[0042] And / or, oxidized polymers and their derivatives are prepared by oxidation of natural polymers, including at least one of hyaluronic acid, sodium alginate, chondroitin sulfate, carboxymethyl cellulose, and dextran;

[0043] And / or, nitrogen-containing polymers and their derivatives include at least one of amination polymers and their derivatives, collagen, ε-polylysine, and chitosan; amination polymers and their derivatives include AG and amination silk fibroin;

[0044] And / or, the secondary cross-linking agent includes at least one of transglutaminase, thrombin, and horseradish peroxidase.

[0045] It should be noted that in some specific embodiments, perfluorohexane (PFH) is preferably used as the acoustic response unit for the following reasons: First, it has higher acoustic response efficiency. Perfluorohexane is a low-boiling-point liquid that can undergo a liquid-gas phase transition. Under relatively low-intensity diagnostic ultrasound, it can undergo violent and instantaneous vaporization, generating microbubbles. This process is accompanied by significant volume expansion and cavitation effects, which can efficiently and thoroughly destroy the liposome structure, ensuring the rapid and complete release of the secondary crosslinking agent. In contrast, existing calcium-loaded liposomes only use ultrasound to create transient pores in the lipid membrane, allowing calcium ions to be passively diffused and released. This approach has higher release efficiency, and the mechanical force generated by the phase transition is stronger and more controllable. Second, it has additional imaging capabilities. The microbubbles generated by the phase transition of perfluorohexane can themselves serve as excellent ultrasound contrast agents, giving the embolization agent built-in imaging capabilities, facilitating real-time monitoring of its distribution, morphology, and embolization effect during treatment. In contrast, calcium ions in existing technologies do not have imaging capabilities, requiring the addition of additional contrast components, which increases the complexity of preparation and potential risks. Third, it has better biocompatibility. Perfluorohexane is chemically inert, has excellent biocompatibility, and is ultimately excreted through respiration, posing no significant toxic side effects to the human body. In contrast, the instantaneous release of large amounts of calcium ions in existing technologies may cause local ion concentration imbalances, posing a potential risk of electrophysiological interference. Furthermore, existing technologies often present transglutaminase in a free form. In embolic agent applications, premature and non-specific release or leakage of this large enzyme in the bloodstream could catalyze unintended cross-linking of proteins at non-target sites, posing a potential biosafety risk. This proposed solution, by encapsulating a secondary cross-linking agent in liposomes, effectively avoids this problem.

[0046] In some embodiments, the ultrasound-responsive liposomes are prepared from phospholipids having a phase transition temperature; and / or, the ultrasound-responsive liposomes are structurally fragile spherical liposomes, and the ultrasound-responsive liposomes include an additive that reduces the cavitation threshold; and / or, the ultrasound-responsive liposomes are loaded with a sonicating agent; and / or, the surface of the ultrasound-responsive liposomes is coupled with ultrasound microbubbles, and the ultrasound microbubbles are encapsulated with a biocompatible gas; and / or, the interior of the ultrasound-responsive liposomes is filled with a biocompatible gas.

[0047] In some embodiments, the phospholipid having a phase transition temperature includes at least one of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and palmitoyloleoylphosphatidylcholine; and / or, the additive for lowering the cavitation threshold includes at least one of cholesterol and a surfactant; and / or, the acoustic sensitizer includes at least one of protoporphyrin and rose red; and / or, the ultrasonic microbubbles include at least one of air and oxygen; and / or, the biocompatible gas includes at least one of air, oxygen, and carbon dioxide. The surfactant may be a biocompatible nonionic surfactant such as the Tween series or the Span series.

[0048] It should be noted that phospholipids with phase transition temperatures include dipalmitoylphosphatidylcholine (DPPC), which has a phase transition temperature of approximately 41°C, making it suitable for the physiological environment in vivo. Additives that lower the cavitation threshold include at least one of cholesterol and surfactants, which can reduce the minimum ultrasonic intensity required for liposomes to exhibit cavitation effects and improve response sensitivity. Acoustic sensitizers include at least one of protoporphyrin and rose red; under ultrasonic irradiation, these sensitizers are activated and generate reactive oxygen species, which can efficiently disrupt the lipid bilayer structure of liposomes and promote the release of secondary cross-linking agents. Ultrasonic microbubbles include air or oxygen; after ultrasonic-responsive liposomes are coupled with ultrasonic microbubbles, the ultrasonic microbubbles oscillate and cavitate under ultrasonic irradiation, and the resulting mechanical force can further disrupt the liposome structure, ensuring the full release of secondary cross-linking agents.

[0049] In some embodiments, nonionic contrast agents and / or particulate contrast agents are also included; nonionic contrast agents include at least one of iopamidol, iodixanol, iopromide, and iohexol; particulate contrast agents include at least one of barium sulfate micron particles, tantalum nanoparticles, and liquid metal; when particulate contrast agents are mixed into the raw materials of hydrogel embolization agents for formulation, they need to be uniformly dispersed by an ultrasonic disruptor.

[0050] It should be noted that the hydrogel embolization agent of the present invention, by adding iohexol powder, has endogenous imaging visualization function, which can monitor the embolization effect in real time, help doctors assess the embolization range, and enhance the accuracy and safety of treatment.

[0051] In some embodiments, the preparation method of oxidized polymers and their derivatives is as follows: dissolve oxidized polymers and their derivatives in water, add an oxidant to react in the dark, and after the reaction is terminated, dialyze and freeze dry to obtain oxidized polymers and their derivatives.

[0052] In some embodiments, the preparation method of nitrogen-containing polymers and their derivatives is as follows: dissolve the polymers and their derivatives in a buffer solution, add an amination reagent and an activating solution to react, adjust the pH to a preset range, and after the reaction, dialyze and freeze dry to obtain the amination polymers and their derivatives.

[0053] In some embodiments, the method for preparing ultrasound-responsive liposomes is as follows: phospholipids and their derivatives are dissolved in an organic solvent, and a lipid film is obtained by rotary evaporation under reduced pressure. A suspension is obtained by elution with a secondary crosslinking agent solution. Phase change droplets are added to the suspension and mixed evenly. After centrifugation, washing, and resuspension, ultrasound-responsive liposomes loaded with secondary crosslinking agent and phase change droplets are obtained.

[0054] This embodiment provides a method for preparing an ultrasound-triggered enzymatic cross-linked hydrogel embolizer, comprising the following steps: mixing oxidized polymers and their derivatives, and nitrogen-containing polymers and their derivatives with solvents respectively to obtain solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives; mixing the solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives with ultrasound-responsive liposomes to obtain an ultrasound-triggered enzymatic cross-linked hydrogel embolizer.

[0055] The applicant further provides the following specific embodiments to describe the present invention. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0056] Example 1

[0057] This embodiment provides a method for preparing an ultrasound-triggered enzymatic cross-linked hydrogel embolization agent, comprising the following steps:

[0058] Step S1: Preparation of oxidized hyaluronic acid (OHA), amination gelatin (AG), and ultrasound-responsive liposomes:

[0059] Preparation of OHA: Dissolve 5g of sodium hyaluronate in 500mL of deionized water, add 50mL of 0.05mol / L sodium periodate solution, and react at room temperature in the dark for 6h; after the reaction is completed, add 1mL of ethylene glycol, and continue stirring for 30min to terminate the oxidation reaction, to obtain the first solution; transfer the first solution to a dialysis bag and dialyze for 3-5 days, and freeze-dry the dialyzed first solution to obtain a light yellow or white flocculent OHA powder;

[0060] Preparation of AG: Weigh 5g of gelatin and add it to 50mM MES buffer. Heat and stir at 40-50℃ until the gelatin is completely dissolved, then cool to room temperature. Add 15g of adipate dihydrazide (ADH) and stir until the ADH is completely dissolved to obtain a second mixture. Dissolve 13.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 9.0g of 1-hydroxybenzotriazole (HOBt) in DMSO solvent (DMSO and water volume ratio 1:1), and heat or sonicate as needed. To aid dissolution, an activating solution is obtained, which must be used within 15 minutes. The activating solution is then slowly added dropwise to the second mixture, with continuous stirring to ensure uniform reaction. The pH is adjusted to 5.0 ± 0.2 with dilute hydrochloric acid or NaOH solution, and the mixture is reacted at room temperature for 24 hours to obtain the third solution. The third solution is transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 8–14 kDa and dialyzed for 3–5 days, with deionized water replaced every 6–8 hours. The dialysis product is freeze-dried to obtain a white AG powder, which is then dried and stored for later use.

[0061] Preparation of ultrasound-responsive liposomes: 8 mg dipalmitoylphosphatidylcholine (DPPC), 4 mg distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 4 mg cholesterol were dissolved in 10 mL of chloroform, then transferred to a 100 mL pear-shaped flask. The organic solvent was removed by rotary evaporation under reduced pressure at 45 °C, forming a uniform lipid film on the flask wall. The lipid film was eluted with 5 mL of a 10 mg / mL transglutaminase (TGase) solution to obtain a translucent suspension. The suspension was then... Add 50 μL of perfluorohexane (PFH) dropwise and treat with an ultrasonic cell disruptor for 5 min under ice bath conditions (120 W, 5 s operation / 5 s interval) to obtain the fourth solution; centrifuge the fourth solution at 20,000 × g for 30 min, discard the supernatant, wash the precipitate 3 times, and resuspend the precipitate in sterile PBS to obtain TGase-encapsulated ultrasonic-responsive liposomes (hereinafter referred to as LTP); under the same preparation conditions, without adding PFH and TGase enzyme, prepare blank liposomes (hereinafter referred to as LiP) and store them at 4 °C for later use;

[0062] Step S2, preparing the hydrogel embolization agent: Prepare a 5% (w / w) OHA solution and a 10% (w / w) AG solution. Since the densities of OHA and AG are extremely close to those of water, a 5% (v / v) OHA solution and a 10% (v / v) AG solution can also be prepared. Add ultrasound-responsive liposomes to the OHA solution, with the volume of the ultrasound-responsive liposomes being 1 / 6 of the OHA volume. Add iohexol to the AG solution, with the mass fraction of iohexol in the AG solution being 30%. Quickly mix the OHA solution and AG solution at a volume ratio of 3:7 using a three-way valve to obtain the hydrogel embolization agent. In the hydrogel embolization agent, the volume fractions of OHA, AG, and ultrasound-responsive liposomes are 1.5%, 7%, and 1.17%, respectively.

[0063] The OHA, AG, ultrasound-responsive liposomes, and hydrogel embolizing agents prepared in Example 1 were characterized. The specific test methods and results are as follows:

[0064] Test Example 1: Morphology and stability testing of ultrasound-responsive liposomes

[0065] Test methods: 1) The particle size distribution and zeta potential of LTP and LiP were quantitatively characterized using a Malvern particle size analyzer to verify the quality of LTP preparation. Particle size analysis was performed according to ISO 22412:2017 standard, and potential analysis was performed according to GB / Z42353-2023 standard; 2) The specific steps for liposome stability testing are as follows: LTP and LiP were placed at room temperature and stored for one week. After one week, the appearance changes of the two liposomes were first observed by visual inspection, focusing on abnormal phenomena such as color change, transparency change and precipitation. Then, the particle size distribution and potential values ​​of the two liposomes were tested again using a Malvern particle size analyzer. The parameter changes before and after storage were compared to comprehensively evaluate the long-term storage stability of the liposome carrier.

[0066] Test results: Figure 2 The figures show the morphology and related performance characteristics of LTP and Lip; among them, Figure 2 In the diagram, A represents a comparison of the particle size distribution of the two types of liposomes. Figure 2 B in the diagram represents the potential comparison. Figure 2 C in the figure represents a comparison of the appearance stability of the two liposomes after one week. Figure 2 D in the figure represents a comparison of the intensity distribution of the two types of liposomes after one week. Figure 2 E in the figure represents a comparison of the potential changes of the two types of liposomes after one week.

[0067] Figure 2 As shown in A, the average particle size of Lip is 142 nm and the average particle size of LTP is 165 nm. The particle size distribution of both liposomes is relatively uniform, indicating that the prepared liposomes have suitable size and good dispersibility. Figure 2 In B, the average Zeta potentials of Lip and LTP were -2.73 mV and -3.41 mV, respectively, indicating that both liposomes had negative charges on their surfaces. Furthermore, the successful loading of TGase and PFH did not significantly affect the surface charge of the liposomes. This potential characteristic can effectively prevent the aggregation of liposomes in vivo and ensure their dispersion stability. Figure 2 C~ Figure 2 The E value indicates that after LTP and Lip were left to stand at room temperature for one week, neither showed any obvious abnormalities in appearance: no color change, no precipitation, and no significant change in transparency. Furthermore, the particle size distribution and potential value of LTP did not change significantly compared to before standing, and its intensity and intensity change trend were not significantly different from those of Lip, confirming that LTP has good long-term stability.

[0068] Test Example 2: In vitro ultrasound imaging effect of ultrasound-responsive liposomes

[0069] Test Method: A 2% agarose solution was prepared and solidified at low temperature to form an in vitro ultrasound model. The in vitro ultrasound imaging effect of ultrasound-responsive liposomes was evaluated using an ultrasound imaging system. Using the commercial ultrasound contrast agent SonoVe as a control, LTP solution and SonoVe solution were injected into the in vitro simulation model, respectively. The model was irradiated with ultrasound equipment. The response under different ultrasound parameters was monitored and recorded in real time during the experiment, and the ultrasound imaging effects of the two were compared.

[0070] Test results: Figure 3 This image compares the in vitro ultrasound imaging effects of LTP and the commercial ultrasound contrast agent SonoVe. Under the same ultrasound irradiation conditions, the ultrasound imaging effect produced by LTP is highly comparable to that of the commercial contrast agent SonoVe. Specifically, under ultrasound stimulation, LTP significantly enhances the intensity of the ultrasound imaging signal, resulting in clear imaging and stable signals, effectively achieving in vitro ultrasound visualization. Experimental results confirm that LTP possesses excellent ultrasound imaging capabilities.

[0071] Test Example 3: Comparative Test of Modification Characterization of Hydrogel Matrix

[0072] Test methods: This test focuses on the characterization experiments of the hydrogel matrix (amined gelatin AG, oxidized hyaluronic acid OHA) and precursor hydrogel. The specific test methods and reference standards are as follows: 1) Verification of the necessity of modification: Unmodified gelatin (hereinafter referred to as Gel), unmodified sodium hyaluronate (hereinafter referred to as HA), and AG and OHA prepared in this application were used to conduct hydrogel synthesis experiments at 37℃ to observe whether the system can form a stable hydrogel matrix and to verify the necessity of modification by gelatin and sodium hyaluronate; 2) Flow 2 3) Fourier transform infrared spectroscopy (FTIR) test: In accordance with GB / T6040-2019 standard, FTIR tests were performed on Gel, AG, HA and OHA respectively to analyze the changes in functional groups of the materials before and after modification and to verify whether the modification reaction was successful; 4) One-H nuclear magnetic resonance spectroscopy (¹H-NMR) test: In accordance with GB / T21189-2007 standard, ¹H-NMR tests were performed on Gel, AG, HA and OHA respectively to further confirm the success of the modification reaction and the introduction of functional groups through changes in characteristic peaks.

[0073] Test results: Figure 4 The figures show a comparison of the characterization of modified hydrogel matrices; among them, Figure 4 In the figure, A represents the characterization of the curing properties of hydrogels prepared by gel and HA at simulated human physiological temperature; Figure 4 B-1 in the figure is a comparison of the FTIR spectra of Gel and AG; Figure 4 C-1 in the figure represents the ¹H-NMR spectrum of Gel and AG; Figure 4 B-2 in the figure is a comparison of the FTIR spectra of OHA and HA; Figure 4C-2 in the figure represents the ¹H-NMR spectra of OHA and HA.

[0074] Depend on Figure 4 As shown in Figure A, under the condition of 37℃ (simulating the physiological temperature of the human body), the mixture of gel and HA maintained good fluidity and did not form any hydrogel structure. This indicates that unmodified gelatin and sodium hyaluronate cannot form a stable hydrogel matrix under the target conditions, further confirming the necessity of chemically modifying both. Figure 4 As shown in B-1, compared to gel, AG exhibits stronger characteristic absorption peaks in the amide A and amide B segments. This characteristic change confirms that the amino group has been successfully introduced into the gelatin molecule structure, and the degree of amination is high, indicating that the amination modification reaction of gelatin has been successfully completed. Figure 4 As shown in C-1, the ¹H-NMR spectroscopy results further corroborate the success of the modification reaction: Two new characteristic peaks, not present in the Gel spectrum, appeared in the ¹H-NMR spectrum of AG. These new peaks correspond to the characteristic chemical shifts of the amino group, which, along with the FTIR results, further confirms the successful introduction of the amino group. For the characterization results of OHA, see: [See C-1]. Figure 4 B-2 in the sample, compared with HA, has an FTIR spectrum at 1730 cm⁻¹. -1 The characteristic absorption peak of the carbonyl group (C=O) at the position showed no significant change in intensity or shift; however, the ¹H-NMR spectrum (see...) Figure 4 The C-2 data shows that a typical aldehyde characteristic peak appeared in the chemical shift range of 4.7~5.2 ppm. This characteristic peak is direct evidence of the introduction of aldehyde groups after the oxidation of sodium hyaluronate, confirming that sodium hyaluronate has been successfully oxidized to OHA and that the introduction of aldehyde groups has been effective.

[0075] Test Example 4: Evaluation of the rheological properties, injectability, and enzymatic crosslinking properties of hydrogel embolic agents

[0076] Test methods: 1) Injectability test: Using a 20G standard needle, the hydrogel embolization agent was injected to observe the smoothness of the injection process and whether any blockage occurred, thus evaluating its injectability; 2) Rheological property test: Referring to YY / T 1435-2016 standard, a rheometer was used to test the relationship between the fluidity and shear rate of the hydrogel embolization agent and its strain cycle performance, evaluating the shear thinning characteristics and structural recovery ability of the hydrogel embolization agent; at the same time, the change in the storage modulus (G') of the hydrogel embolization agent before and after ultrasonic stimulation was tested to evaluate the enzymatic secondary crosslinking effect under ultrasonic triggering; 3) Fourier transform infrared spectroscopy (FTIR) test: Referring to GB / T 6040-2019 standard, the crosslinking reaction changes were further verified; 1H nuclear magnetic resonance spectroscopy (¹H-NMR) test: Referring to GB / T 21189-2007 standard, the functional group reaction characteristics were further verified.

[0077] Test results: Figure 4 D in the diagram represents the injectability properties of the hydrogel embolization agent. Figure 4 E in the figure represents the relationship between the flowability of the hydrogel embolization agent and the shear rate. Figure 4 In this context, F represents the result of a strain cyclic experiment; Figure 4 G in the figure represents the change in storage modulus of the hydrogel embolization agent under ultrasonic stimulation.

[0078] Figure 4 The "D" indicates that the hydrogel embolization agent can be successfully injected through a 20G needle. The injection process is smooth and there is no blockage or jamming, indicating that the hydrogel embolization agent has good fluidity and injectability, which can meet the injection needs in clinical embolization treatment. Figure 4 E in the figure represents the relationship between the flowability of the hydrogel embolization agent and the shear rate, showing a positive correlation between the two. When the shear strain changes from 0% to 500%, the hydrogel embolization agent exhibits obvious shear thinning characteristics: when an external force (shear action) is applied, the viscosity of the hydrogel embolization agent decreases and it flows. After the external force is removed, the viscosity can quickly return to its original state. This characteristic ensures smooth injection of the hydrogel embolization agent and rapid molding after injection. Figure 4 The strain cycle test results provided by F show that in the alternating strain cycle test of 500% and 1%, the hydrogel embolizer can still recover to the original gel state after 3 cycles without obvious structural damage. This further confirms that the hydrogel embolizer has excellent shear thinning ability and structural recovery performance, which not only helps the fluidity during the injection process, but also enables it to quickly recover the gel structure after injection and exert the embolizing effect. Figure 4 The graph shows the change in storage modulus (G') of the TGase-containing hydrogel embolizer before and after ultrasonic stimulation. After ultrasonic stimulation, the storage modulus (G') of the hydrogel embolizer rapidly increased from 100 Pa to 1087 Pa, indicating that the mechanical properties of the hydrogel embolizer were significantly enhanced. Figure 4 The value of G indicates that after ultrasound stimulation, the storage modulus (G') of the hydrogel embolization agent rapidly increased from 100 Pa to 1087 Pa, indicating a significant enhancement in its mechanical properties. This phenomenon demonstrates that under ultrasound irradiation, the glutamine transaminase (TGase) released by the liposomes can further promote the secondary cross-linking reaction within the hydrogel embolization agent through a catalytic reaction, thereby significantly improving its mechanical strength and meeting the requirements for material mechanical properties in embolization therapy.

[0079] Test Example 5: Injection Force and Imaging Ability Test of Hydrogel Embolizing Agents

[0080] Test methods: 1) Injection force test: The push rod end of a 1mL screw-type syringe is precisely contacted with the upper pressure plate of the electronic universal testing machine, and the lower end of the syringe is vertically and stably fixed on the fixture of the testing machine; three clinically commonly used microcatheters of different specifications, namely 2.4-F, 2.5-F and 4-F, are connected respectively, and 1mL of hydrogel embolizing agent is added into the syringe to ensure uniform filling and no air bubbles; the upper pressure plate is set to advance at a uniform speed of 1mL / min, and the pressure data of the upper pressure plate during the advancement is recorded in real time; to ensure the reliability and repeatability of the experimental data, each group of experiments is repeated 3 times, and the average value of the 3 test data is taken as the final injection force result; 2) Imaging ability test: Hydrogel embolizing agents containing different mass concentrations of iohexol are prepared and imaging detection is performed using a CT scanner; the CT scanning parameters are set as follows: tube voltage 80~120kV, tube current 50~100mA, slice thickness 0.5~1.0mm, pitch ≈1.0, to ensure that the scanning parameters are uniform and conform to the clinical routine testing standards. Using CT image analysis software, the CT values ​​of hydrogel embolization agents at different iohexol concentrations were accurately measured. Combined with image clarity, the imaging effect of hydrogel embolization agents was comprehensively evaluated. At the same time, supplementary experiments were carried out in an in vitro simulated environment and subcutaneously in rats to compare the ultrasound imaging capabilities of hydrogel embolization agents with those of the commercial ultrasound contrast agent SonoVe.

[0081] Test results: Figure 5 This image shows an injection force testing device and imaging effect characterization diagram for hydrogel embolic agents; among which, Figure 5 In the diagram, A represents the injection force testing device. Figure 5 In the image, B represents the X-ray and ultrasound imaging effects of hydrogel embolization agents with different concentrations of iohexol. Figure 5 C in the figure is a comparison of the ultrasound imaging effects of the hydrogel embolization agent and SonoVe (including in vitro and subcutaneous rat experiments).

[0082] According to generally accepted clinical standards, the injection force of embolic agents should be less than 50N to reduce the workload on physicians and ensure a smooth injection process. Figure 5 As shown in A, regardless of the ratio of AG solution to OHA solution, the injection force is less than 5N, indicating that this hydrogel embolizer has excellent injection performance in routine clinical microcatheter procedures and can easily complete the injection operation. In particular, when using a 2.7-F microcatheter (a commonly used specification for delicate clinical procedures), the hydrogel embolizer with a volume ratio of 7:3 for AG solution to OHA solution has the optimal injection force, approximately 18.9N, which is far below the clinical standard threshold (50N) and fully meets the ideal standard for embolizer injection. Therefore, all subsequent tests used a volume ratio of 7:3 for AG solution to OHA solution to further optimize the clinical injection characteristics of the hydrogel embolizer. Figure 5As shown in Figure B, the addition of iohexol can effectively improve the imaging effect of hydrogel embolization agents. The imaging effect is optimal when the mass concentration of iohexol in the hydrogel embolization agent is 30%. The specific location, morphology and diffusion range of the hydrogel embolization agent can be clearly displayed in both X-ray and ultrasound images, without blurring or artifacts. This provides clinicians with an intuitive and effective real-time monitoring method, facilitating precise control of the embolization process. Figure 5 As indicated by C in the figure, the results of in vitro and subcutaneous supplementation experiments in rats show that the ultrasound imaging ability of this hydrogel embolization agent is comparable to that of the commercial ultrasound contrast agent SonoVe. Under ultrasound examination, it can present a clear and stable imaging signal, further verifying the reliability of the imaging ability and clinical applicability of the hydrogel embolization agent.

[0083] Test Example 6: Biocompatibility and Coagulation Capacity Test of Hydrogel Embolizing Agents

[0084] Test methods: 1) Cytotoxicity test: Referring to ISO 10993-5:2009, the CCK8 cell viability assay combined with live / dead cell staining was used to evaluate the effects of hydrogel embolization on cell growth, viability, and morphology; different concentrations of hydrogel embolization extract were set up, with a focus on testing the cytotoxicity at a high concentration (25 mg / mL) to verify the biocompatibility and safety of the hydrogel embolization; 2) Hemolysis test: Referring to ISO 10993-4:2017, the interaction between the hydrogel embolization and blood was tested. The hemolysis rate after contact with the liquid was assessed to evaluate its blood compatibility and determine whether it would cause a significant hemolytic reaction, ensuring the safety of contact with blood in clinical applications; 3) Coagulation ability test: Prepare recalcified whole blood (mix 0.1M calcium chloride solution with citrate blood at a volume ratio of 1:10), add 1mL of recalcified whole blood to an EP tube, and then add 200μL of hydrogel embolizing agent. The time required for the formation of a firm blood clot was observed and recorded in real time using the inverted method. The coagulation time was used as the evaluation index to assess the coagulation promoting ability of the hydrogel embolizing agent.

[0085] Test results: Figure 6 The graphs represent the biocompatibility and coagulation capacity of the hydrogel embolization agent; among them, Figure 6 Figure A in the graph shows the cell viability detection results under different concentrations of hydrogel embolizing agent extract. Figure 6 In the image, B represents the staining effect of live and dead cells. Figure 6 C in the diagram represents the hemolysis rate of the hydrogel embolization agent; Figure 6 D in the figure represents a comparison of the coagulation ability of hydrogel embolic agents.

[0086] Figure 6Figures A and B show that even with a concentration of up to 25 mg / mL (far exceeding the concentration used in clinical practice), the cell viability remained above 98%, and no obvious abnormalities in cell morphology were observed through live and dead cell staining (no cell shrinkage, rupture, or other toxic manifestations). This indicates that the hydrogel embolizer can maintain excellent biocompatibility under high concentration conditions, does not produce significant toxicity to cells, and will not affect the growth of normal human cells. Figure 6 The hemolysis test results shown in C indicate that the hemolysis rate of the hydrogel embolizer after contact with blood is less than 5%, which meets the hemolysis safety standard for medical biomaterials. This shows that when it comes into contact with blood in clinical applications, it will not cause significant hemolytic reactions, will not damage red blood cells, and will not cause blood-related adverse reactions, further confirming its blood compatibility and safety as a medical embolization material. Figure 6 The coagulation ability test results shown in Figure D indicate that when the hydrogel embolization agent is added to recalcified whole blood, it significantly shortens the time for the formation of a strong blood clot, and the coagulation efficiency is significantly improved compared with the blank control group. This result demonstrates that the hydrogel embolization agent possesses excellent coagulation-promoting capabilities, accelerating the blood coagulation process and facilitating the rapid formation of a stable blood clot at the embolization site, thereby improving the embolization effect and further enhancing its clinical application performance as a medical embolization agent.

[0087] Test Example 7: Rabbit Kidney Embolism Model Testing with Hydrogel Embolizing Agent

[0088] Test Methods: This test used a New Zealand rabbit to construct an in vivo model of renal embolism, focusing on the in vivo embolization procedure, multimodal follow-up monitoring, and pathological evaluation of the hydrogel embolization agent. Specific test methods, operating procedures, and detection standards are as follows:

[0089] (1) Experimental grouping: Based on the follow-up time points, the experimental rabbits were divided into a control group and experimental groups on day 7, day 14 and day 28. The number of experimental rabbits in each group was n=3 to ensure that the experimental data were statistically significant and reproducible.

[0090] (2) Animal anesthesia and preoperative preparation: All experimental New Zealand rabbits were anesthetized with a combination of Sutamaxin 50 (5mg / kg, intramuscular injection) and Sutamaxin (2mg / kg, intramuscular injection) to ensure stable anesthesia effect and appropriate depth of anesthesia. The experimental rabbits were fasted and deprived of water for 24 hours before the operation to avoid gastrointestinal contents affecting the surgical field and postoperative recovery.

[0091] (3) Surgical area preparation: The left groin area of ​​the experimental rabbit was prepared, disinfected with povidone-iodine, and covered with a sterile surgical drape. The principle of aseptic operation was strictly followed to prevent surgical site infection.

[0092] (4) Femoral artery separation and treatment: The skin, muscles and fascia of the groin area are cut layer by layer, and the femoral artery is separated by blunt dissection. The distal end of the femoral artery is ligated, and the proximal end is temporarily clamped with an arterial clamp. 2-0 sutures are reserved for use to avoid arterial bleeding during the operation.

[0093] (5) Microcatheter insertion and fixation: Use microscissors to cut open the anterior wall of the femoral artery, immediately insert a 2.7-F microcatheter, fix it with the reserved 2-0 suture by tying a slipknot, loosen the arterial clamp, and continue to advance the microcatheter to the appropriate position.

[0094] (6) Selective renal artery catheterization and angiography: Under the guidance of the microguidewire and real-time X-ray fluoroscopy monitoring, the microcatheter is selectively inserted into the right renal artery; after the microguidewire is withdrawn, renal artery angiography is performed to confirm the patency of the right renal artery and the accuracy of the catheterization position, so as to ensure that the embolization operation is precisely targeted to the right renal artery.

[0095] (7) Hydrogel embolization injection and ultrasound triggering: Under strict X-ray fluoroscopy monitoring, the hydrogel embolization agent (AG solution and OHA solution in a volume ratio of 7:3, containing 30% iohexol) was slowly injected. During the injection, the contrast agent reflux, ectopic embolization and other abnormalities were closely observed to ensure the safety of embolization. After the injection was completed, the liposomes were triggered to burst using an ultrasound diagnostic instrument, and ultrasound imaging was achieved at the same time to enhance the embolization effect and verify the reliability of imaging.

[0096] (8) Postoperative management: After the embolization was completed, renal artery angiography was performed to confirm the embolization effect; then the microcatheter was removed, the surgical incision was sutured layer by layer, and the incision was bandaged and fixed with sterile gauze; for the first 3 days after the operation, 800,000 units of penicillin were injected intramuscularly daily to prevent infection of the surgical incision and the body, and the mental state, diet and activity of the experimental rabbits were closely observed after the operation.

[0097] (9) Postoperative follow-up and testing: On the 14th and 28th day after surgery, the experimental rabbits in the experimental group underwent CT, enhanced CT, routine ultrasound and contrast-enhanced ultrasound examinations. At the same time, on the embolization procedure, the 7th, 14th and 28th day after surgery, all experimental rabbits underwent abdominal CT scans. Blood was collected from the marginal ear vein to test blood routine and biochemical indicators, and to assess the overall physiological status and liver and kidney function of the experimental rabbits. After the follow-up, the experimental rabbits were euthanized, and the right kidney, renal artery and important organs such as heart, liver, lungs and brain were removed and fixed in formalin for 24 hours for subsequent pathological morphological evaluation.

[0098] (10) Specific testing methods:

[0099] CT examination method: A CT scanner was used, and the scanning parameters were set as follows: tube voltage 120kV, tube current 80mA, slice thickness 0.625mm. Iohexol (300mgI / mL, 2mL / kg) was injected into the marginal ear vein before scanning. CT images were acquired at 12 seconds (arterial phase) and 70 seconds (parenchymal phase) after injection. The images of both kidneys were processed using three-dimensional reconstruction technology to clearly observe the morphology and blood flow changes of the kidneys.

[0100] Ultrasound examination method: First, in the conventional ultrasound mode, the size and shape of the kidney are measured and the outline of the kidney is observed; then, the color Doppler mode is switched to detect the distribution of blood flow signals inside the kidney; finally, the ultrasound contrast mode is switched to, and the contrast microbubbles generated by the PFH contained in the embolization agent or the commercial contrast agent SonoVe under the action of ultrasound are used to record real-time dynamic video for 90 seconds to evaluate the blood flow blockage of the kidney after embolization and the contrast effect.

[0101] Test results: Figure 7 Imaging and related characterization images of a rabbit kidney embolization model using hydrogel embolization agents; among them, Figure 7 In the image, A represents the ultrasound-triggered liposome rupture and imaging effect. Figure 7 B in the diagram represents the location of the microcatheter and the injection path of the hydrogel embolization agent under X-ray fluoroscopy. Figure 7 C in the image represents a comparison of DSA angiography and Doppler ultrasound blood flow signals before and after embolization. Figure 7 D in the image represents a CT scan of vital organs 28 days post-surgery.

[0102] In the experiment, a catheter was inserted via the femoral artery, superselectively positioned in the right renal aorta, and 1 mL of hydrogel embolic agent was injected for embolization. Subsequently, ultrasound stimulation was used to trigger a secondary cross-linking reaction of the hydrogel embolic agent, further enhancing the embolization effect. See [link to relevant documentation]. Figure 7 In section A, firstly, digital subtraction angiography (DSA) was used to monitor the situation before, during, and after embolization. Before embolization, DSA images clearly showed that both kidneys were of normal size and position, with no abnormalities observed. During embolization, Figure 7 In Figure B, the white arrow indicates the location of the catheter (4F catheter), while the red arrow indicates the flow path of the hydrogel embolization agent. The hydrogel embolization agent is being slowly injected into the right renal artery through the 4F catheter. After embolization, we performed DSA imaging again, and the results showed that the right kidney was not visible on the image, proving that the embolization was successful and the blood flow to the right kidney had been effectively blocked. See [link to DSA image]. Figure 7In the study, rabbits underwent multimodal imaging follow-up to evaluate the stability and safety of the hydrogel embolization agent. CT and enhanced CT imaging techniques revealed a significant reduction in the volume of the right kidney without enhancement, indicating complete blood flow occlusion and stable embolization. Furthermore, CT reconstruction clearly showed that the embolized right kidney was no longer visible, further confirming the success of the embolization. To further verify the precision of the embolization process and the stability of the hydrogel embolization agent, Doppler ultrasound, micro-flow imaging, and contrast-enhanced ultrasound were performed. The results showed no blood flow signal in the right kidney, further demonstrating the successful embolization of the right renal artery by the hydrogel embolization agent without any reperfusion. (See [reference needed]). Figure 7 C in [the original text]. See also [the original text]. Figure 7 In the case of D, 28 days after embolization, a follow-up CT scan was performed. No obvious abnormalities were observed in the rabbit's heart, liver, lungs, brain and other important organs. The hydrogel embolization agent did not migrate to other organs.

[0103] The above are merely preferred embodiments of this application and are not intended to limit the present invention. Although this application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the foregoing examples or make equivalent substitutions for some of the technical features. However, 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 ultrasound-triggered enzymatic cross-linking hydrogel embolization agent, characterized in that, Including oxidized polymers and their derivatives, nitrogen-containing polymers and their derivatives, ultrasound-responsive liposomes, and solvents; The oxidized polymer and its derivatives, and the nitrogen-containing polymer and its derivatives undergo a first cross-linking and solidification process via Schiff base reaction to form a hydrogel matrix. The ultrasound-responsive liposomes are spherical liposomes, and the interior of the spherical liposomes is filled with a secondary crosslinking agent and phase change droplets; The phase change droplets are used to receive ultrasonic signals and change phase to gaseous state to destroy the structure of the spherical liposomes and release the secondary crosslinking agent; the secondary crosslinking agent is used to react with the residual active groups in the hydrogel matrix to achieve the second crosslinking and curing of the hydrogel matrix.

2. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 1, characterized in that, In the enzyme-crosslinked hydrogel embolizing agent, the volume fraction of oxidized polymers and their derivatives is 1-2%, the volume fraction of nitrogen-containing polymers and their derivatives is 5-10%, and the volume fraction of ultrasound-responsive liposomes is 1-2%.

3. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 1, characterized in that: The phase change droplets include at least one of perfluorohexane and perfluoropentane; And / or, the oxidized polymer and its derivatives are prepared by oxidation of natural polymers, wherein the natural polymers include at least one of hyaluronic acid, sodium alginate, chondroitin sulfate, carboxymethyl cellulose, and dextran; And / or, the nitrogen-containing polymers and their derivatives include at least one of amination polymers and their derivatives, collagen, ε-polylysine, and chitosan; And / or, the secondary cross-linking agent includes at least one of transglutaminase, thrombin, and horseradish peroxidase.

4. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 3, characterized in that, The ultrasound-responsive liposomes are prepared from phospholipids having a phase transition temperature; and / or, the ultrasound-responsive liposomes include an additive that lowers the cavitation threshold; and / or, the ultrasound-responsive liposomes are loaded with a sonicating agent; and / or, the surface of the ultrasound-responsive liposomes is coupled with ultrasound microbubbles, which are encapsulated with a biocompatible gas; and / or, the interior of the ultrasound-responsive liposomes is filled with a biocompatible gas.

5. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 4, characterized in that: The phospholipid with phase transition temperature includes at least one of dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and palmitoyloleoylphosphatidylcholine; and / or, the additive that lowers the cavitation threshold includes at least one of cholesterol and surfactant; and / or, the sonication agent includes at least one of protoporphyrin and rose red; the biocompatible gas includes at least one of air, oxygen, and carbon dioxide.

6. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 1, characterized in that, It also includes nonionic contrast agents and / or particulate contrast agents; The nonionic contrast agent includes at least one of iopamidol, iodixanol, iopromide, and iohexol; The particulate developer includes at least one of barium sulfate micron particles, tantalum nanoparticles, and liquid metal.

7. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 1, characterized in that, The preparation method of the oxidized polymer and its derivatives is as follows: Oxidized polymers and their derivatives are dissolved in water, and an oxidizing agent is added to react in the dark. After the reaction is terminated, the polymers are dialyzed and freeze-dried to obtain oxidized polymers and their derivatives.

8. The ultrasound-triggered enzymatic cross-linking hydrogel embolizer according to claim 3, characterized in that, The preparation method of the amination polymer and its derivatives is as follows: The polymer and its derivatives were dissolved in a buffer solution, and an amination reagent and activating solution were added to react. After adjusting the pH to a preset range, the reaction was carried out by dialysis and freeze-drying to obtain the amination polymer and its derivatives.

9. The ultrasound-triggered enzymatic cross-linking hydrogel embolization agent according to claim 1, characterized in that, The method for preparing the ultrasound-responsive liposomes is as follows: Phospholipids and their derivatives are dissolved in an organic solvent and subjected to rotary evaporation under reduced pressure to obtain a lipid film. The film is then eluted with a secondary crosslinking agent solution to obtain a suspension. Phase change droplets are added to the suspension and mixed evenly. After centrifugation, washing, and resuspension, ultrasonically responsive liposomes loaded with secondary crosslinking agent and phase change droplets are obtained.

10. A method for preparing an ultrasound-triggered enzymatically cross-linked hydrogel embolizing agent according to any one of claims 1 to 9, characterized in that, Includes the following steps: Oxidized polymers and their derivatives, and nitrogen-containing polymers and their derivatives are respectively mixed with solvents to obtain solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives; the solutions of oxidized polymers and their derivatives and solutions of nitrogen-containing polymers and their derivatives are mixed with ultrasound-responsive liposomes to obtain an ultrasound-triggered enzyme-crosslinked hydrogel embolization agent.

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