Preparation method of self-developing embolism microspheres

Abstract

The invention belongs to the technical field related to tumors, and discloses a preparation method of self-developing embolism microspheres. Comprising the following steps: producing calcium alginate microspheres through cross-linking, obtaining polyelectrolyte hydrogel microspheres through electrostatic complexation, and obtaining the self-developing embolism microspheres through amino cross-linking. According to the invention, sodium alginate and polyamino acid are used as raw materials for preparation, so that the preservative is safe and non-toxic and has biodegradability, and the degradation product has no toxic or side effect on human body. The microspheres are prepared by adopting an electrostatic spraying method, organic solvents are not used, and the microspheres are green and environment-friendly and have no toxic residues.

Description

Technical Field

[0001] This invention belongs to the field of tumor-related technology, specifically relating to a method for preparing autoradiographic embolization microspheres that can be used for interventional therapy. Background Technology

[0002] Transcatheter arterial embolization (TAE) is the preferred palliative treatment for advanced liver cancer and other malignant diseases. Under imaging guidance, embolic materials are injected into the tumor's feeding artery, blocking blood supply to "starve" the tumor and simultaneously releasing drugs locally to kill it. It offers advantages such as being minimally invasive, safe, and targeted, leading to its increasingly widespread application. Embolization microspheres have attracted considerable attention due to their high targeting specific tissues and organs, good embolization effect, compatibility with chemotherapy drugs, and sustained drug release capabilities, making them one of the most common embolization carriers currently available. Clinically used embolization microspheres mainly include polyvinyl alcohol (PVA), polylactic acid-glycolic acid copolymer (PLGA), and sodium alginate microspheres. While these microspheres offer advantages such as good biocompatibility and embolization efficacy, they also have drawbacks, including the inability to visualize themselves. They cannot be tracked or located in vivo and can only be visualized by mixing with exogenous contrast agents. Therefore, it is impossible to accurately determine the distribution of microspheres in blood vessels and the final actual location of the embolization. Furthermore, the location of the embolic agent cannot be reported in real time during the procedure, which greatly inconveniences the surgical operation, affects the accuracy of determining the embolization endpoint, and thus impacts efficacy evaluation. It also increases the risk of embolization at non-target sites. Therefore, endowing microspheres with self-visible imaging capabilities, making them visible in blood vessels and tissues, is particularly important. This would significantly reduce the amount of contrast agent used, thereby reducing allergic reactions and potential nephrotoxicity. Moreover, X-ray detection equipment can directly observe the location of the embolization microspheres in blood vessels, improving the efficiency and safety of embolization.

[0003] Advances in materials science and preparation technology have made the development of self-illuminating embolic microspheres possible. For example, combining contrast agents such as barium sulfate, tantalum nanoparticles, and iodized oil with embolic materials such as alginate can produce self-illuminating embolic microspheres, enabling direct visualization of the location and distribution of embolic agents within blood vessels without the need for additional contrast agents. However, microspheres prepared using physical embedding methods also have drawbacks such as drug exudation and aggregation / precipitation. Leakage of the contrast agent can cause toxic side effects on patients, such as thrombosis caused by insoluble solids, edema and nausea caused by iodine-based contrast agents, and allergic reactions. Microspheres prepared by chemical cross-linking methods, due to the introduction of iodine-containing compounds into their structure, exhibit more stable chemical properties and better biocompatibility, and are gradually attracting attention in the industry. Currently, most self-illuminating microspheres prepared by chemical methods use synthetic polymers, organic solvents, and cross-linking agents. Furthermore, the selection of carrier materials and the control of experimental temperature and solution concentration involve complex preparation processes, thus requiring consideration of toxic residues. Additionally, the microspheres are non-degradable, and long-term retention in the body can also have adverse effects on patients. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method for preparing self-illuminating embolization microspheres. Polyelectrolyte hydrogel microspheres are prepared using biomaterials such as sodium alginate and polyamino acids as raw materials. Iodine-containing L-thyroxine is grafted onto polyamino acid molecules through the cross-linking effect of genipin, giving it X-ray imaging capability. The microspheres are blue and visible to the naked eye.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: a method for preparing autoradiographic embolic microspheres, comprising the following steps:

[0006] (1) Sodium alginate was dissolved in purified water to obtain an aqueous solution of a certain concentration. This solution was then added dropwise to a CaCl2 solution using an electrostatic spray method, and the alginate reacted with CaCl2... 2+ Cross-linking yielded calcium alginate microspheres of different diameters;

[0007] (2) The above calcium alginate microspheres were added to polylysine solution for reaction, and polyelectrolyte hydrogel microspheres were obtained through electrostatic complexation of carboxyl and amino groups.

[0008] (3) The above-mentioned polyelectrolyte hydrogel microspheres were added to a mixed solution of genipin and thyroxine for reaction. Thyroxine was grafted onto the amino group of polyamino acids through the amino cross-linking effect of genipin, resulting in blue microspheres with X-ray autoradiography function.

[0009] Furthermore, in step (1), the sodium alginate has a molecular weight of 100–300 kDa and a solution concentration of 1.0–3.0 wt%.

[0010] Furthermore, in step (2), the polyamino acid is polylysine, polyornithine, or a mixture of the two, the concentration of the reaction solution is 0.1 to 2.0 wt%, the volume ratio of microspheres to solution is 1:5 to 30, and the reaction time is 10 to 40 min.

[0011] Furthermore, in step (3), the concentration of thyroxine in the mixed solution is 0.1–1.0 wt%, the concentration of genipin is 0.1–1.0 wt%, the volume ratio of microspheres to solution is 1:5–30, the reaction temperature is 37–45 °C, and the reaction time is 1–4 h.

[0012] Furthermore, the thyroxine in step (3) can be L-thyroxine or D-thyroxine, or its sodium salt, or can be replaced with 3,3′,5-triiodothyronine, iodoacetamide, iodoaniline, or a compound containing iodine and amino groups.

[0013] Another objective of this invention is to protect the autoradiographic embolism microspheres prepared by the above-described preparation method, which have a diameter of 50–800 μm.

[0014] Another object of the present invention is to protect the application of the above-mentioned self-illuminating embolization microspheres in embolization materials.

[0015] The advantages of this invention compared to the prior art are:

[0016] 1. The method for preparing self-illuminating embolic microspheres provided by this invention uses sodium alginate and polyamino acids as raw materials. It is safe, non-toxic, and biodegradable, with degradation products having no toxic side effects on the human body. The microspheres are prepared using an electrostatic spraying method, without the use of organic solvents, making it green and environmentally friendly, and leaving no toxic residues.

[0017] 2. The self-illuminating embolization microsphere preparation method provided by this invention grafts thyroxine onto polyamino acid molecules through genipin amino cross-linking. Since the thyroxine molecule contains four iodine atoms, the microspheres possess X-ray self-illuminating capabilities. Because the iodine atoms are grafted onto the material molecules rather than embedded within the microspheres, they exhibit advantages such as chemical stability and excellent biocompatibility, and will not negatively impact patients due to contrast agent leakage.

[0018] 3. The self-illuminating embolization microsphere preparation method provided by the present invention uses genipin, a natural biological cross-linking agent, for cross-linking. It is safe and non-toxic, has good biocompatibility, and the generated product is blue. Therefore, the prepared microspheres are visible to the naked eye, which is convenient for clinical embolization treatment.

[0019] 4. The self-developing embolization microsphere preparation method provided by the present invention has mild reaction conditions, does not involve high temperature and high pressure reaction conditions, and does not generate heat during the reaction process. There is no risk of safety accidents such as explosion or leakage. The operation is stable and controllable, and it is easy to carry out large-scale production. Attached Figure Description

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 Example 1: Schematic diagram of products prepared from calcium alginate microspheres of different particle sizes;

[0022] Figure 2 Example 2: Schematic diagram of the product prepared from sodium alginate-polylysine polyelectrolyte microspheres;

[0023] Figure 3 Example 3: Schematic diagram of the product prepared by X-ray autoradiography microspheres;

[0024] Figure 4 Example 4: Results of rabbit arterial embolization experiment. Detailed Implementation

[0025] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0026] Example 1: Preparation of calcium alginate microspheres of different particle sizes by electrostatic spraying

[0027] 1. Preparation steps

[0028] (1) Sodium alginate was dissolved in purified water to obtain aqueous solutions of different concentrations. These solutions were then added dropwise to a CaCl2 solution using an electrostatic spray method and reacted for 30 minutes. The carboxyl groups in the sodium alginate molecule reacted with the CaCl2... 2+ Cross-linking yielded calcium alginate microspheres of different diameters;

[0029] (2) Under an inverted microscope, five fields of view were randomly selected to obtain no less than 100 images of microspheres. The particle size of each microsphere was read using ImageJ software, and the average value was calculated as D0.

[0030] 2. Relationship between preparation conditions and microsphere size

[0031] Sodium alginate concentration (wt%) Microsphere size (μm) 1 1.0 101.2±15.7 2 1.2 158.4±16.3 3 1.5 262.6±20.9 4 2.0 385.2±19.1

[0032] The above experimental results show that the diameter of calcium alginate microspheres is directly related to the concentration of sodium alginate; the microsphere diameter increases with increasing sodium alginate concentration. (See attached...) Figure 1 It can be seen that the microspheres have good sphericity and particle size uniformity, indicating that the prepared microspheres are of good quality.

[0033] Example 2: Preparation of calcium alginate-polylysine polyelectrolyte microspheres

[0034] 1. Preparation steps

[0035] (1) Sodium alginate was dissolved in purified water to obtain a 1.5 wt% aqueous solution. This solution was then added dropwise into a CaCl2 solution using an electrostatic spray method and reacted for 30 minutes. The carboxyl groups in the sodium alginate molecule reacted with the CaCl2... 2+ Cross-linking yielded calcium alginate microspheres of different diameters;

[0036] (2) The above calcium alginate microspheres were added to polylysine solutions of different concentrations and reacted for 20 min. Polyelectrolyte microspheres were obtained through electrostatic complexation of carboxyl and amino groups.

[0037] (2) Under an inverted microscope, five fields of view were randomly selected to obtain no less than 100 images of microspheres. The particle size of each microsphere was read using ImageJ software, and the average value was calculated as D0.

[0038] 2. Measurement of microsphere settling time and accumulation volume

[0039] Add 1 ml of microspheres to a 1 ml graduated cylinder. Start timing immediately after adding the microspheres. Stop timing when the total volume of the microspheres in the graduated cylinder stops changing. Read the settling time and measure the accumulated volume.

[0040] 3. Relationship between polylysine concentration and microsphere performance

[0041]

[0042] As can be seen from the table above, the electrostatic complexation reaction between calcium alginate microspheres and polylysine can make the microsphere structure more compact, reduce the volume and diameter of the microspheres, and cause the surface of the microspheres to wrinkle. Figure 2 The microsphere shrinkage rate is positively correlated with the polylysine concentration. This is mainly because the electrostatic force between the amino and carboxyl groups makes the microsphere structure more compact, resulting in a decrease in microsphere diameter. Increasing the amount of polylysine increases the degree of electrostatic complexation, thus leading to a greater degree of microsphere shrinkage. However, when the polylysine concentration exceeds 0.4 wt%, further increases in concentration result in less volume shrinkage. This may be because the carboxyl groups in the calcium alginate gel have likely already completely reacted with the amino groups, and further increases in the amount of amino groups do not improve the degree of reaction. The microsphere stacking volume also decreases with increasing polylysine concentration, indicating that increasing the degree of reaction makes the microspheres more compact, which is beneficial for improving the embolization treatment effect. The microsphere settling time is between 4 and 6 minutes, which is relatively fast and conducive to rapid vascular occlusion in clinical practice. The settling time is positively correlated with the microsphere diameter; the smaller the microsphere diameter, the longer the settling time, but the difference in settling time is not significant.

[0043] Example 3: Preparation of X-ray Autoradiography Microspheres

[0044] 1. Preparation steps

[0045] (1) Sodium alginate was dissolved in purified water to obtain a 1.2 wt% aqueous solution. This solution was then added dropwise to a CaCl2 solution using an electrostatic spray method, and the alginate reacted with CaCl2... 2+ Cross-linking yielded calcium alginate microspheres of different diameters;

[0046] (2) The above calcium alginate microspheres were added to a 0.2% polylysine solution for reaction, and polyelectrolyte microspheres were obtained through electrostatic complexation of carboxyl and amino groups.

[0047] (3) The above polyelectrolyte microspheres were added to a mixed solution of genipin and thyroxine of different concentrations for reaction. Through the cross-linking effect of genipin, thyroxine was grafted onto the amino group of the polyamino acid to obtain blue microspheres with X-ray autoradiography function.

[0048] 2. In vitro autoradiography performance of embolization microspheres

[0049] Calcium alginate and autoradiating microspheres were placed in a 24-well plate, and X-rays were irradiated at a height of 1000 μm above the liquid surface using an X-ray machine to observe the development of the microspheres.

[0050] 3. Experimental Results

[0051] from Figure 3 It can be seen that the microspheres are blue due to the cross-linking effect of genipin, and the color intensity is positively correlated with the concentration of genipin. When the concentration of genipin reaches 0.4%, the microspheres are no longer transparent, which helps to enhance the clinical X-ray imaging signal. Figure 3 The X-ray imaging ability of the microspheres was also investigated. Calcium alginate microspheres did not exhibit autoradiographic activity, while autoradiographic microspheres, due to the presence of iodine, had strong imaging activity.

[0052] Example 4 Rabbit Arterial Embolization Experiment

[0053] 1. Experimental Methods

[0054] (1) Preparation of experimental animals

[0055] After weighing the Japanese white rabbits, anesthetize them with 1 ml of sodium pentobarbital (0.4%) per kg of body weight, fix them on a rabbit board, and remove the hair and skin from the throat area for later use.

[0056] (2) Percutaneous embolization

[0057] The animal's laryngeal turbinates are bluntly dissected layer by layer in the midline to expose the left common carotid artery. The internal and external carotid arteries are dissected along the blood flow direction, and the proximal end of the common carotid artery is closed with an arterial clamp. A suture is threaded through the external carotid artery for future surgical manipulation; a PVC cannula is also prepared. Autoradiographic microspheres (1 ml / kg) soaked in physiological saline are injected into the common carotid artery through the external carotid artery. The external carotid artery is ligated, the cannula is carefully removed, the wound is ligated, the arterial clamp is released, and the wound is cleaned and sutured.

[0058] 2. Study on embolization effect

[0059] Figure 4 These are X-ray images of animals at different postoperative periods. The arrows indicate the embolization sites of the microspheres, and the different imaging conditions at different times can be clearly observed. As can be seen from the image, the difference between preoperative (A) and postoperative (B) is very obvious: the target site was difficult to distinguish before surgery, while a white imaging area appeared after surgery—representing the location of the target blood vessel. This autoradiography function can directly mark the lesion tissue and also has the effect of blocking blood flow. Figure 4C and D in the images are radiographic images taken 4 and 8 weeks after embolization, respectively. The white area indicates the presence of autoradiographic microspheres, meaning that the embolization effect of the microsphere embolization agent can be maintained for more than 8 weeks. This result shows that this microsphere preparation with autoradiographic function can fully meet the clinical requirements for the localization and observation of patient tissues and postoperative follow-up.

[0060] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing self-illuminating embolic microspheres, characterized in that the steps include... include: S1. Sodium alginate was dissolved in purified water to obtain an aqueous solution of a certain concentration. This solution was then added dropwise to a CaCl2 solution using an electrostatic spray method, and the alginate reacted with CaCl2... 2+ Cross-linking yielded calcium alginate microspheres of different diameters; S2. The above-mentioned calcium alginate microspheres were added to a polylysine solution for reaction, and polyelectrolyte hydrogel microspheres were obtained through electrostatic complexation of carboxyl and amino groups. S3. The above-mentioned polyelectrolyte hydrogel microspheres are added to a mixed solution of genipin and thyroxine for reaction. Through the amino cross-linking effect of genipin, thyroxine is grafted onto the amino group of the polyamino acid to obtain blue microspheres with X-ray autoradiography function.

2. The method for preparing self-illuminating embolic microspheres according to claim 1, characterized in that, In step S1, the sodium alginate has a molecular weight of 100–300 kDa and a solution concentration of 1.0–3.0 wt%.

3. The method for preparing self-illuminating embolic microspheres according to claim 1, characterized in that, In step S2, the polyamino acid is polylysine, polyornithine, or a mixture of the two. The concentration of the reaction solution is 0.1–2.0 wt%, the volume ratio of microspheres to solution is 1:5–30, and the reaction time is 10–40 min.

4. The method for preparing self-illuminating embolic microspheres according to claim 1, characterized in that, In step S3, the concentration of thyroxine in the mixed solution is 0.1–1.0 wt%, the concentration of genipin is 0.1–1.0 wt%, the volume ratio of microspheres to solution is 1:5–30, the reaction temperature is 37–45 °C, and the reaction time is 1–4 h.

5. The method for preparing self-illuminating embolic microspheres according to claim 1, characterized in that, The thyroxine in step S3 can be L-thyroxine or D-thyroxine, or its sodium salt, or can be replaced with 3,3′,5-triiodothyronine, iodoacetamide, iodoaniline, or a compound containing iodine and amino groups.

6. The self-illuminating embolic microspheres prepared by the method of claim 1, characterized in that, The diameter is 50–800 μm.

7. The application of the self-illuminating embolic microspheres as described in claim 6 in embolic materials.

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