Injectable developing embolism microsphere preparation based on hydroxyapatite as well as preparation method and application of injectable developing embolism microsphere preparation

Abstract

The invention discloses an injectable developing embolism microsphere preparation based on hydroxyapatite and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that hydroxyapatite microspheres loaded with an iodine contrast agent are prepared from hydroxyapatite microspheres and an iodine contrast agent injection; preparing HA1-iodine contrast agent-hydroxyapatite microspheres by using the hydroxyapatite microspheres loaded with the iodine contrast agent and large-molecular-weight hyaluronic acid HA1, mixing the HA1-iodine contrast agent-hydroxyapatite microspheres with small-molecular-weight hyaluronic acid HA2 to obtain mixed dry powder, mixing the mixed dry powder with a liquid carrier, and performing ultrasonic dispersion to obtain the iodine contrast agent-hydroxyapatite microspheres loaded with the iodine contrast agent. The injectable developing embolism microsphere preparation based on hydroxyapatite can achieve radiography under DSA and CT, slow sedimentation can be achieved in a specified blood vessel local part so as to achieve the uniform embolism effect, the excellent blood vessel embolism capacity is shown, and the injectable developing embolism microsphere preparation based on hydroxyapatite has wide application prospects in the aspect of interventional embolism treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an injectable developing embolic microsphere preparation based on hydroxyapatite, and a preparation method and application thereof. Background Art

[0002] Transvascular interventional therapy is a commonly used minimally invasive treatment method in clinical practice. Transcatheter Arterial Embolization (TAE) has become the preferred treatment method for advanced liver cancer. TAE technology accurately delivers embolic materials to the target blood vessels through a catheter, thereby achieving effective embolization of the blood vessels to achieve the established treatment goals. This technology has the advantages of minimal invasiveness, real-time imaging monitoring throughout the process, and highly selective target blood vessel cannulation, which greatly improves the accuracy and safety of embolization operations. Through TAE, doctors can implement precise treatment for a variety of diseases such as tumors, vascular malformations, hemorrhage, hypersplenism, etc., effectively curb the progression of the disease, and at the same time protect the surrounding normal tissues from damage to the greatest extent. The selection and quality of embolic agents is one of the most critical factors in TAE treatment. The embolic materials currently used in clinical practice include small spring steel coils, gelatin sponge particles, iodized oil, polyvinyl alcohol microspheres, etc.

[0003] With the continuous change of TAE technology, a variety of new embolic microspheres such as CalliSpheres, HepaSphere, DC Bead, etc. have benefited patients, and researchers have also developed a variety of embolic microspheres. For example, the Chinese patent document with publication number CN115671317A discloses a multimodal development embolic microsphere and its preparation method and application. The multimodal development embolic microsphere includes an embolic microsphere and a development microsphere loaded on the surface of the embolic microsphere. The embolic microsphere includes at least one of Embosphere, Callisphere or DC bead. The development microsphere includes Fe3O4 particles, gold particles and characteristic Raman molecules. The surface of Fe3O4 particles is positively charged, gold particles are loaded on Fe3O4 particles, and characteristic Raman molecules are loaded on gold particles. The multimodal development embolic microsphere can realize multimodal development under CT, MRI and Raman, and improve the efficacy of embolic therapy. The Chinese patent document with publication number CN114081989A discloses a biodegradable embolic microsphere and a preparation method thereof, wherein the embolic microsphere comprises a microsphere matrix (biodegradable chitosan and hyaluronic acid), a stabilizer, a cross-linking agent (containing carboxyl and amino groups), an oil-soluble solvent and a surfactant, and is prepared by a one-step method using microfluidic technology. The embolic microsphere has good biocompatibility and degradability, and the particle size distribution of the finished microsphere is uniform.

[0004] However, the existing embolic microspheres still have many shortcomings: (1) Currently, embolic microspheres are mainly made of high molecular weight polymers, and their biosafety needs to be improved; (2) Most drug-loaded microspheres cannot be imaged in real time, resulting in the inability to monitor the position of the microspheres during and after surgery, affecting intraoperative judgment and the choice of postoperative treatment plans; (3) The current embolic microspheres settle too quickly, resulting in uneven local deposition in blood vessels, affecting the embolic effect. Therefore, it is necessary to develop a new type of injectable imaging embolic microsphere preparation. Summary of the invention

[0005] The present invention provides a method for preparing an injectable developing embolic microsphere preparation based on hydroxyapatite. The prepared injectable developing embolic microsphere preparation can realize angiography under DSA and CT, and can realize slow sedimentation in a designated blood vessel to achieve a uniform embolic effect, showing excellent vascular embolic ability.

[0006] The specific technical solutions adopted are as follows:

[0007] A method for preparing an injectable developing embolic microsphere preparation based on hydroxyapatite comprises the following steps:

[0008] (1) taking hydroxyapatite microspheres and iodine contrast agent injection, mixing them under negative pressure under stirring, washing, and rotating to obtain hydroxyapatite microspheres loaded with iodine contrast agent; the particle size of the hydroxyapatite microspheres is 80 μm-2000 μm;

[0009] (2) adding iodine contrast agent-loaded hydroxyapatite microspheres to a hyaluronic acid HA1 aqueous solution, and freeze-drying to obtain HA1-iodine contrast agent-hydroxyapatite microspheres, mixing HA1-iodine contrast agent-hydroxyapatite microspheres with hyaluronic acid HA2 powder to obtain a mixed dry powder, and mixing the mixed dry powder with a liquid carrier to obtain the hydroxyapatite-based injectable imaging embolic microsphere preparation;

[0010] The molecular weight of the hyaluronic acid HA1 is 100K Dalton-400K Dalton, and the molecular weight of the hyaluronic acid HA2 is 2K Dalton-5K Dalton.

[0011] Hydroxyapatite is a calcium phosphate-based compound and the main inorganic component of human and animal bones. As an inert material, it has the advantages of being non-toxic, non-irritating, non-allergic, non-mutagenic, non-hemolytic, and non-destructive to biological tissues, and can be imaged under X-ray irradiation. Therefore, the present invention uses hydroxyapatite as a core material, and further loads an iodine contrast agent on the hydroxyapatite, and then coats it with high molecular weight hyaluronic acid HA1, and finally mixes it with low molecular weight hyaluronic acid HA2 to obtain a product preparation, which is easy to inject and use, and can achieve slow sedimentation in a designated blood vessel to achieve a uniform embolization effect.

[0012] Specifically, the hydroxyapatite microspheres have pores, and after negative pressure mixing, the iodine contrast agent is loaded on the pores or surfaces of the hydroxyapatite microspheres.

[0013] Optionally, the iodine contrast agent is at least one of iopromide, iohexol, diatrizoate, sodium diatrizoate, and iodixanol.

[0014] Preferably, the ratio of hydroxyapatite microspheres to iodine contrast agent injection is 1 g: 5-20 mL, and the iodine concentration in the iodine contrast agent injection is 50-400 mg / mL.

[0015] Preferably, the hydroxyapatite microspheres can be specifically selected from one or a combination of two or more specifications of microspheres with a particle size of 80 μm-200 μm, microspheres with a particle size of 120 μm-300 μm, microspheres with a particle size of 300 μm-500 μm, microspheres with a particle size of 500 μm-700 μm, microspheres with a particle size of 800 μm-1200 μm, and microspheres with a particle size of 1200 μm-2000 μm.

[0016] Preferably, the molecular weight of the hyaluronic acid HA1 is 100K Dalton-200K Dalton, and the molecular weight of the hyaluronic acid HA2 is 2K Dalton-3K Dalton.

[0017] Preferably, the concentration of the hyaluronic acid HA1 aqueous solution is 20-100 mg / mL.

[0018] Preferably, in the mixed dry powder, the mass ratio of HA1-iodine contrast agent-hydroxyapatite microspheres to hyaluronic acid HA2 powder is 1:0.075-0.75.

[0019] Preferably, the liquid carrier is an aqueous solution selected from deionized water or a buffer solution; in the injectable developing embolic microsphere preparation based on hydroxyapatite, the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres is 15wt%-40wt%.

[0020] The present invention also provides an injectable developing embolic microsphere preparation based on hydroxyapatite prepared by the preparation method of the injectable developing embolic microsphere preparation based on hydroxyapatite.

[0021] The present invention also provides the use of the injectable developing embolic microsphere preparation based on hydroxyapatite in the preparation of disease treatment and / or monitoring products, wherein the diseases include but are not limited to tumors, vascular malformations, hemorrhage, hypersplenism, etc.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention provides an injectable embolic microsphere preparation based on hydroxyapatite, wherein the core material is hydroxyapatite, the hydroxyapatite is loaded with iodine contrast agent, and the outer layer is wrapped with hyaluronic acid of different molecular weights. The introduction of high molecular weight hyaluronic acid HA1 and low molecular weight hyaluronic acid HA2 makes the microsphere preparation easy to inject and use, and can achieve slow sedimentation in the designated blood vessel to achieve a uniform embolic effect. The injectable embolic microsphere preparation can achieve angiography under DSA and CT.

[0024] (2) The microspheres prepared by the method of the present invention have uniform particle size and controllable size, and the preparation method is simple and low-cost. The large molecular weight hyaluronic acid HA1 coating has a protective effect and a sustained-release effect, and the small molecular weight hyaluronic acid HA2 is convenient for injection as a base. In addition, the raw materials of the injectable developing embolic microsphere preparation of the present invention, such as hydroxyapatite, hyaluronic acid, and iodine contrast agent, are all approved for use in the human body and have good biosafety.

[0025] (3) The hydroxyapatite-based injectable developing embolic microsphere preparation provided by the present invention exhibits excellent vascular embolic ability and good X-ray developing ability in both in vivo and in vitro experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 SEM image of the hydroxyapatite-based injectable embolic microsphere preparation.

[0027] Figure 2 This is a picture of HA1-iodinated contrast agent-hydroxyapatite microspheres under a laser confocal microscope.

[0028] Figure 3 This is the CT imaging of the hydroxyapatite-based injectable embolic microsphere preparation.

[0029] Figure 4 The figure shows the in vitro development of the hydroxyapatite-based injectable embolic microsphere preparation under DSA.

[0030] Figure 5 The image shows the imaging of the hydroxyapatite-based injectable embolic microsphere preparation in the liver under DSA.

[0031] Figure 6 Figure 4: Renal embolization with injectable hydroxyapatite-based embolic microspheres under DSA. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the examples and drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Contents not described in detail in this specification belong to prior art known to professionals and technicians in the field.

[0033] Example 1

[0034] 1) Preparation of HA1 (high molecular weight hyaluronic acid aqueous solution)

[0035] 0.45 g of hyaluronic acid solid powder with a molecular weight of 100K Dalton was accurately weighed, and then 20 ml of deionized water was added to the hyaluronic acid powder to obtain a hyaluronic acid HA1 aqueous solution with a concentration of 0.022 g / ml.

[0036] 2) Hydroxyapatite microspheres loaded with iopromide

[0037] Accurately weigh 9.0g of hydroxyapatite microspheres with a particle size of 80μm-200μm, place it in a 200ml Schlenk bottle, add 60ml iopromide injection (iodine concentration is 370mg / mL) thereto, under magnetic stirring, use a double-row pipe to evacuate the system to negative pressure and maintain negative pressure for 10min, then transfer the resulting mixed turbid liquid to a 50ml centrifuge tube, and use 10ml deionized water to wash the remaining solid. Then the centrifuge tube is fixed on a rotary mixer, and the speed is set to 20r / min and kept for 24h. The resulting turbid liquid uses a centrifuge to centrifuge for 5min at 2300r / min, separates the upper liquid, and obtains the hydroxyapatite microspheres loaded with iopromide.

[0038] 3) Hydroxyapatite microspheres adsorb HA1

[0039] Add 20ml step 1) hyaluronic acid HA1 aqueous solution obtained in the hydroxyapatite microspheres of the load iopromide obtained in the above-mentioned steps, and mix evenly after being vibrated, and keep it for 1h. Gained mixed solution is separated by supernatant liquid using centrifuge with 2300r / min rotating speed centrifuge 5min. Add 20ml deionized water, use centrifuge with 2300r / min rotating speed centrifuge 5min after mixing, separate supernatant liquid, wash away residual hyaluronic acid molecules. Gained precipitate is removed internal moisture using lyophilization, obtains the hydroxyapatite microspheres (HA1-iodine contrast agent-hydroxyapatite microspheres) of the load with iopromide adsorbed HA1.

[0040] 4) Preparation of hydroxyapatite microsphere mixed dry powder

[0041] An analytical balance was used to accurately weigh 3.0 g of HA1-iodine contrast agent-hydroxyapatite microspheres, and 0.7 g of hyaluronic acid solid powder with a molecular weight of 3 kilodaltons was also accurately weighed using an analytical balance. The two were thoroughly shaken and mixed using an oscillator to prepare 3.7 g of mixed dry powder.

[0042] 5) Preparation of injectable embolic microspheres based on hydroxyapatite

[0043] When it is needed, accurately weigh 3.7g of the mixed dry powder, add deionized water thereto, shake thoroughly, and when the low molecular weight hyaluronic acid is dissolved, the components in the system are evenly mixed to obtain an injectable developing embolic microsphere preparation based on hydroxyapatite. Adjust the amount of deionized water added so that the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres in the injectable developing embolic microsphere preparation based on hydroxyapatite is 30wt%.

[0044] Example 2

[0045] The difference between this embodiment and embodiment 1 is only that:

[0046] In step 1), a hyaluronic acid solid powder with a molecular weight of 150K Daltons is used to prepare a hyaluronic acid HA1 aqueous solution with a concentration of 0.022 g / ml;

[0047] In step 2), the particle size of the hydroxyapatite microspheres is 120 μm-300 μm, and the iodine contrast agent injection is iohexol injection (iodine concentration is 140 mg / mL);

[0048] In step 4), a mixed dry powder is prepared using a hyaluronic acid solid powder with a molecular weight of 5K Daltons;

[0049] In step 5), the amount of deionized water added is adjusted so that the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres in the hydroxyapatite-based injectable developing embolic microsphere preparation is 25wt%;

[0050] The rest of the preparation process and parameters are the same as those in Example 1.

[0051] Example 3

[0052] The difference between this embodiment and embodiment 1 is only that:

[0053] In step 1), a hyaluronic acid solid powder with a molecular weight of 200K Daltons is used to prepare a hyaluronic acid HA1 aqueous solution with a concentration of 0.022 g / ml;

[0054] In step 2), the particle size of the hydroxyapatite microspheres is 300 μm-500 μm, and the iodine contrast agent injection is diatrizoate injection (iodine concentration is 200 mg / mL);

[0055] In step 4), a mixed dry powder is prepared using a hyaluronic acid solid powder with a molecular weight of 2K Daltons;

[0056] In step 5), the amount of deionized water added is adjusted so that the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres in the hydroxyapatite-based injectable developing embolic microsphere preparation is 35wt%;

[0057] The rest of the preparation process and parameters are the same as those in Example 1.

[0058] Example 4

[0059] The difference between this embodiment and embodiment 1 is only that:

[0060] In step 2), the iodine contrast agent injection is iodixanol injection (iodine concentration is 270 mg / mL);

[0061] In step 4), a mixed dry powder is prepared using a hyaluronic acid solid powder with a molecular weight of 4K Daltons;

[0062] In step 5), the amount of deionized water added is adjusted so that the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres in the hydroxyapatite-based injectable developing embolic microsphere preparation is 15 wt %;

[0063] The rest of the preparation process and parameters are the same as those in Example 1.

[0064] Example 5

[0065] The difference between this embodiment and embodiment 1 is only that:

[0066] In step 2), the particle size of the hydroxyapatite microspheres is 500 μm-700 μm, and the iodine contrast agent injection is iohexol injection (iodine concentration is 180 mg / mL);

[0067] In step 5), the amount of deionized water added is adjusted so that the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres in the hydroxyapatite-based injectable developing embolic microsphere preparation is 15 wt %;

[0068] The rest of the preparation process and parameters are the same as those in Example 1.

[0069] Sample analysis

[0070] Figure 1 This is a SEM picture of the hydroxyapatite-based injectable developing embolic microsphere preparation prepared in Example 1 after drying. The microspheres are spherical or quasi-spherical, and hyaluronic acid can be seen covering the surface of the microspheres. The pores are filled, proving that the surface of the microspheres is wrapped with hyaluronic acid.

[0071] Figure 2This is a picture of HA1-iodine contrast agent-hydroxyapatite microspheres (HAP-MS) (parameters in Example 1, high molecular weight hyaluronic acid HA1 is labeled with FITC, HA-FITC) under a laser confocal microscope. The hydroxyapatite microspheres loaded with iodine contrast agent are mixed with high molecular weight hyaluronic acid HA1 linked to FITC, proving that the high molecular weight hyaluronic acid HA1 is successfully adsorbed onto the hydroxyapatite surface.

[0072] The amount of deionized water added was adjusted so that the mass fractions of HA1-iodine contrast agent-hydroxyapatite microspheres in the injectable hydroxyapatite-based embolic microsphere preparation of Example 1 were 30wt%, 25wt%, 20wt%, 15wt%, 10wt%, 5wt%, and 0wt% (deionized water control), respectively. Figure 3 The corresponding CT images show that the CT values ​​of the injectable embolic microsphere preparations based on hydroxyapatite with different mass fractions under CT are significantly higher than that of the pure water group, and the preparations can be developed under CT.

[0073] Figure 4 This is the in vitro development image of the hydroxyapatite-based injectable embolic microsphere preparation under DSA in Example 1. It can be seen that the microsphere preparation group on the right has a better development effect under DSA than the pure water on the left.

[0074] Figure 5 This is the imaging image of the hydroxyapatite-based injectable imaging embolic microsphere preparation in the liver under DSA of Example 1. The hydroxyapatite-based injectable imaging embolic microsphere preparation was injected into the pig liver, and obvious imaging was observed locally in the pig liver.

[0075] Figure 6 This is a picture of kidney embolization using the injectable hydroxyapatite-based embolic microsphere preparation under DSA in Example 1. It can be seen that the renal artery is truncated, proving that the renal blood vessels are successfully embolized.

[0076] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an injectable developing embolic microsphere preparation based on hydroxyapatite, characterized in that: The following steps are involved: (1) taking hydroxyapatite microspheres and iodine contrast agent injection, mixing them under negative pressure under stirring to obtain hydroxyapatite microspheres loaded with iodine contrast agent; the particle size of the hydroxyapatite microspheres is 80 μm-2000 μm; (2) adding iodine contrast agent-loaded hydroxyapatite microspheres to a hyaluronic acid HA1 aqueous solution, and freeze-drying to obtain HA1-iodine contrast agent-hydroxyapatite microspheres, mixing HA1-iodine contrast agent-hydroxyapatite microspheres with hyaluronic acid HA2 powder to obtain a mixed dry powder, and mixing the mixed dry powder with a liquid carrier to obtain the hydroxyapatite-based injectable imaging embolic microsphere preparation; The molecular weight of the hyaluronic acid HA1 is 100K Dalton-400K Dalton, and the molecular weight of the hyaluronic acid HA2 is 2K Dalton-5K Dalton.

2. The method for preparing the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 1, characterized in that: The iodine contrast agent is at least one of iopromide, iohexol, diatrizoate, sodium diatrizoate and iodixanol.

3. The method for preparing the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 1, characterized in that: The ratio of hydroxyapatite microspheres to iodine contrast agent injection is 1 g: 5-20 mL, and the iodine concentration in the iodine contrast agent injection is 50-400 mg / mL.

4. The method for preparing the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 1, characterized in that: The concentration of the hyaluronic acid HA1 aqueous solution is 20-100 mg / mL.

5. The method for preparing the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 1, characterized in that: In the mixed dry powder, the mass ratio of HA1-iodine contrast agent-hydroxyapatite microspheres to hyaluronic acid HA2 powder is 1:0.075-0.

75.

6. The method for preparing the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 1, characterized in that: The liquid carrier is an aqueous solution selected from deionized water or a buffer solution; in the injectable developing embolic microsphere preparation based on hydroxyapatite, the mass fraction of HA1-iodine contrast agent-hydroxyapatite microspheres is 15wt%-40wt%.

7. The injectable hydroxyapatite-based embolic microsphere preparation prepared according to the method for preparing the injectable hydroxyapatite-based embolic microsphere preparation according to any one of claims 1 to 6.

8. Use of the hydroxyapatite-based injectable developing embolic microsphere preparation according to claim 7 in the preparation of disease treatment and / or monitoring products.

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