Developable embolizing microspheres and method for preparing the same

Abstract

This invention discloses a method for preparing radiopaque embolic microspheres, belonging to the field of interventional medical device technology. The method includes: S1 preparing an iodine-containing and biodegradable core seed through precipitation polymerization; S2 synthesizing polyvinyl alcohol modified with sulfonic acid groups as a drug-loaded shell material; S3 using a water-in-water emulsion technique to encapsulate the core seed within the shell material, and then thermally crosslinking and curing to form core-shell microspheres; S4 post-processing to obtain the finished product. This invention uses an aqueous system throughout, making it green and safe. The resulting microspheres spatially decouple radiopaqueness and drug-loaded function, solving the risks of radiopaque components interfering with mechanical and drug-loaded properties and permanent foreign body residue in traditional radiopaque microspheres. It has the advantages of clear intraoperative radiopaqueness, a biodegradable core, a soft shell, and high drug loading capacity.

Description

Technical Field

[0001] This invention belongs to the field of interventional medical device technology, specifically relating to a radiopaque embolic microsphere and its preparation method. Background Technology

[0002] Transcatheter arterial embolization (TAE) and chemoembolization (CEE) are important minimally invasive procedures for treating various solid tumors, including hepatocellular carcinoma. To enable real-time intraoperative observation of the location and distribution of microspheres and prevent ectopic embolization, contrast-enhanced embolized microspheres have become a research hotspot. Currently, there are two main approaches to achieving contrast enhancement: one is to physically mix iodized oil or iodine-containing small molecules into the microsphere matrix, but the contrast agent is prone to rapid detachment in vivo, resulting in short-lasting imaging and toxic risks; the other is to covalently bind iodine-containing monomers to the polymer network via chemical bonds. While this solves the detachment problem, it introduces new challenges—extremely high iodine content is required to achieve sufficient CT contrast, leading to microsphere hardening and decreased elasticity, affecting catheter delivery and conformal embolization of the target vessel; simultaneously, the iodine-containing aromatic ring structure "dilutes" the drug-carrying functional groups, resulting in a significant reduction in the chemotherapy drug loading capacity; more critically, chemically bonded iodine does not degrade in vivo for a long time, and the long-term retention of permanent foreign bodies poses long-term biosafety risks, and the permanent high-density images continuously interfere with CT follow-up examinations.

[0003] Therefore, there is an urgent need to develop a new preparation method that can separate the development and drug loading functions, and enable the development components to be safely degraded and metabolized. Summary of the Invention

[0004] In view of this, the present invention aims to provide a method for preparing developable embolic microspheres. The method of the present invention decouples the development and drug-loading functions spatially by first preparing a degradable, developable core seed and then constructing a functionalized drug-loaded shell coating, and uses an aqueous system throughout the process, avoiding the use of organic solvents.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing radiopaque embolic microspheres, comprising the following preparation steps: S1. Dissolve 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate and sebacic acid diacrylate in an ethanol / water mixed solvent, add polyvinylpyrrolidone, and add potassium persulfate under inert gas protection to carry out precipitation polymerization reaction. After the reaction is completed, centrifuge, wash, dialyze and dry to obtain degradable developing core seeds. S2. Polyvinyl alcohol is dissolved in water, activated with an alkaline substance, reacted with 1,3-propanesulfonate lactone, neutralized, dialyzed, and dried to obtain polyvinyl alcohol modified with sulfonic acid groups. S3. A biodegradable developing core seed, polyvinyl alcohol modified with sulfonic acid groups, citric acid, catalyst, and water are mixed and dispersed to obtain a dispersed phase; a polyethylene glycol aqueous solution is used as a continuous phase; the dispersed phase is added to the continuous phase under stirring to form a water-in-water emulsion; then, heating is used to cause the polyvinyl alcohol modified with sulfonic acid groups inside the dispersed phase droplets to undergo esterification, cross-linking, and curing to obtain crude core-shell microspheres. S4. The crude core-shell microspheres are diluted, demulsified, washed, and sieved to obtain the biodegradable and developable core-shell embolic microspheres.

[0006] Preferably, the mass ratio of 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate, sebacic acid diacrylate, ethanol / water mixed solvent, polyvinylpyrrolidone, and potassium persulfate in S1 is 1:(0.2-0.5):100:(0.3-2):(0.01-0.03).

[0007] Preferably, the temperature of the precipitation polymerization reaction in S1 is 60-75℃, and the reaction time is 6-10 hours.

[0008] Preferably, the volume ratio of ethanol to water in the ethanol / water mixed solvent in S1 is 1:(0.8-1.5).

[0009] Free radical copolymerization of 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate and sebacic acid diacrylate was carried out in the presence of potassium persulfate as an initiator. As the polymer chain grew and the molecular weight increased to a certain extent, nuclei precipitated from the ethanol / water mixed solvent, forming micro / nano-sized particles. Polyvinylpyrrolidone was adsorbed onto the particle surface through steric hindrance, preventing particle aggregation and thus obtaining monodisperse core seeds with uniform particle size.

[0010] The sebacic acid diacrylate molecule contains two hydrolyzable ester bonds, allowing the cross-linked network to gradually break down in the aqueous environment and under the action of esterases in vivo. Traditional cross-linking agents such as N,N'-methylenebisacrylamide contain bio-inert amide bonds and are almost non-degradable; however, this invention uses a cross-linking agent containing ester bonds, making the entire imaging core biodegradable in vivo and eliminating the risk of permanent foreign bodies.

[0011] Preferably, the mass ratio of polyvinyl alcohol to 1,3-propanesulfonic acid lactone in S2 is 1:(0.3-0.6).

[0012] Preferably, the degree of alcoholysis of the polyvinyl alcohol in S2 is 85%-99%, and the molecular weight is 5×10⁻⁶. 4 -15×10 4 .

[0013] Preferably, the alkaline substance in S2 is sodium hydroxide.

[0014] Preferably, the reaction temperature in S2 is 20-40℃ and the reaction time is 8-16 hours.

[0015] Preferably, the sulfonic acid group grafting density of the polyvinyl alcohol modified with sulfonic acid groups obtained in S2 is 2.0-4.0 mmol / g.

[0016] This invention utilizes the ring-opening nucleophilic substitution reaction of 1,3-propanesulfonic acid lactone on the hydroxyl groups of polyvinyl alcohol (PVA) under alkaline conditions, introducing sulfonic acid groups into the PVA side chain via stable ether bonds. Sodium hydroxide abstracts protons from the hydroxyl groups of PVA, converting them into more nucleophilic sodium alkoxides, significantly improving the reaction efficiency with 1,3-propanesulfonic acid lactone. Compared to traditional carboxylic acid groups, sulfonic acid groups have lower pKa values, are almost completely ionized under physiological pH conditions, and exhibit stronger electrostatic binding capacity and more stable ion exchange behavior for positively charged chemotherapeutic drugs. This ensures efficient drug loading and controlled sustained release even with the introduction of fewer acidic groups into the shell.

[0017] Preferably, the mass ratio of the degradable developing core seed, sulfonic acid group-modified polyvinyl alcohol, citric acid, catalyst, and water in S3 is (10-40):100:(15-35):(1.5-2.5):(800-1000).

[0018] Preferably, the mass concentration of the polyethylene glycol aqueous solution in S3 is 20-40%, and the molecular weight of the polyethylene glycol is 8000-35000.

[0019] Preferably, the volume ratio of the dispersed phase to the continuous phase in S3 is 1:(5-20).

[0020] Preferably, the stirring temperature in S3 is 30-60℃ and the stirring speed is 200-800 rpm.

[0021] Preferably, the esterification crosslinking curing temperature in S3 is 70-90℃, and the time is 2-8 hours.

[0022] The present invention also provides radiopaque embolic microspheres prepared by the above-described preparation method.

[0023] It contains at least the following beneficial technical effects: This invention first constructs an independent core seed, then encapsulates it in situ with a shell, achieving complete separation of imaging and drug-carrying functions. The shell is made of pure sulfonated polyvinyl alcohol, and the drug loading capacity is unaffected by iodine content, reaching 40-60 mg / mL, with performance comparable to mainstream non-imaging drug-loaded microspheres. The imaging core is copolymerized from ester-bonded monomers and cross-linking agents, which can be gradually degraded into small molecules and metabolized and excreted in vivo, with a designed half-life of 3-6 months. This overcomes the risk of long-term retention as a permanent foreign body in traditional chemically bonded iodine microspheres, and the imaging disappears over time, without affecting subsequent CT follow-up examinations. The rigid iodine-containing polymer is confined within a micron-sized core, and the overall mechanical properties of the microsphere are provided by the high-water-content, flexible shell, resulting in good compressibility and allowing for smooth delivery and conformal embolization through microcatheters. The entire process uses an aqueous system, completely eliminating organic solvents such as liquid paraffin and ethyl acetate, leaving no harmful solvent residues and ensuring high biosafety. By simply adjusting the amount of core seed fed in step S3, the total iodine content of the final microspheres (50-200 mg / g) can be linearly adjusted, which can minimize metal hardening artifacts while ensuring clear intraoperative imaging. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0030] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0031] Example 1 S1. Preparation of biodegradable developing core seeds 0.5 g of polyvinylpyrrolidone was dissolved in 100 mL of an ethanol / water mixture (ethanol to water volume ratio 1:1). 3.0 g of 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate and 1.2 g of sebacic acid diacrylate were added, and the mixture was stirred until dissolved, then purged with nitrogen for 30 min. The temperature was raised to 65 °C, and 0.06 g of potassium persulfate (pre-dissolved in 2 mL of water) was added under nitrogen protection. The reaction was allowed to proceed for 6 h. An additional 0.03 g of potassium persulfate was added, and the temperature was raised to 75 °C for another 2 h. After cooling, the mixture was centrifuged at 12000 rpm for 15 min, washed three times with ethanol / water (1:1) and twice with deionized water, then dispersed and dialyzed in a dialysis bag with a molecular weight cutoff of 14000 for 48 h. The resulting seeds were lyophilized to obtain core seeds with a particle size of 1-2 μm and an iodine content of approximately 400 mg / g.

[0032] S2. Preparation of polyvinyl alcohol modified with sulfonic acid groups 5.0g of polyvinyl alcohol (88% degree of alcoholysis, molecular weight 8.5×10⁻⁶) was added. 4 Dissolve 2.0 g of 1,3-propanesulfonic acid lactone in 100 mL of deionized water (stirring at 90 °C for 2 h) and cool to room temperature. Activate with 2 mL of 1 mol / L NaOH solution for 30 min. Dissolve 2.0 g of 1,3-propanesulfonic acid lactone in 10 mL of tetrahydrofuran and slowly add it dropwise (25-30 °C), reacting for 12 h. Neutralize with 1 mol / L hydrochloric acid to pH≈7, dialyze through a dialysis bag with a molecular weight cutoff of 3500 for 3 days, and lyophilize to obtain sulfonated polyvinyl alcohol with a sulfonic acid group grafting density of 3.2 mmol / g.

[0033] S3. Construction of core-shell microspheres using water-in-water emulsions Dispersed phase: Dissolve 1.0g of sulfonated polyvinyl alcohol in 9.0g of deionized water (dissolve at 90℃ and cool to 40℃), add 0.25g of citric acid and 0.05g of sodium hypophosphite, dissolve, add 200mg of core seeds, and ultrasonically disperse for 20min.

[0034] Continuous phase: 60g of polyethylene glycol (molecular weight 20000) was dissolved in 140g of deionized water to obtain 200mL of 30% solution.

[0035] The continuous phase was heated to 40℃, and the dispersed phase was injected at 1 mL / min with stirring at 400 rpm. After the injection was completed, stirring was continued for 30 min. The temperature was then increased to 85℃ at 1℃ / min and held for 6 h to complete the esterification, crosslinking and curing.

[0036] S4. Post-processing After cooling, pour the mixture into 1L of cold water to break the emulsion. The microspheres will settle and be washed with water until the supernatant is no longer viscous. Sieve the microspheres sequentially through 300μm, 100μm, and 40μm sieves, and collect the 40-100μm and 100-300μm sizes, respectively. Store the microspheres in PBS (pH 7.4).

[0037] Example 2 The only difference from Example 1 is that the amount of sebacic acid diacrylate in S1 is reduced to 0.6g. The remaining steps and parameters are the same as in Example 1.

[0038] Example 3 The only difference from Example 1 is that the amount of sebacic acid diacrylate in S1 is increased to 1.5g. Everything else is the same as in Example 1.

[0039] Example 4 The only difference from Example 1 was that the amount of 1,3-propanesulfonic acid lactone in S2 was adjusted to 1.5 g, and the reaction time was 8 h. The sulfonic acid group grafting density of the obtained sulfonated polyvinyl alcohol was 2.1 mmol / g. The final doxorubicin loading of the microspheres was approximately 42 mg / mL, which was still sufficient to meet the requirements for chemoembolization.

[0040] Example 5 The only difference from Example 1 is that the amount of 1,3-propanesulfonic acid lactone in S2 was adjusted to 3.0 g, the reaction time was 16 h, and the temperature was controlled at 30-35 °C. The resulting grafting density reached 3.9 mmol / g.

[0041] Example 6 The only difference from Example 1 is that in S3, the amount of core seed used is 100mg, citric acid 0.35g, and sodium hypophosphite 0.0875g.

[0042] Example 7 The only difference from Example 1 is that the amount of core seed used in S3 is 400mg and citric acid is 0.15g.

[0043] Example 8 The only difference from Example 1 is that in S3, the volume ratio of the dispersed phase to the continuous phase is adjusted to 1:5, the emulsification temperature is 60°C, the stirring speed is 800 rpm, and the crosslinking temperature is 70°C for 8 hours.

[0044] Comparative Example 1 The only difference from Example 1 is that in S1, equimolar amounts of N,N'-methylenebisacrylamide are used instead of sebacate diacrylate.

[0045] Comparative Example 2 The only difference from Example 1 is that S2 is omitted, and unmodified polyvinyl alcohol is used directly for S3.

[0046] Comparative Example 3 The only difference from Example 1 is that in S3, liquid paraffin (containing 4% Span 80) is used as the continuous phase to form a W / O emulsion, which is then washed with ethyl acetate to remove oil after crosslinking.

[0047] Comparative Example 4 Iodine-containing monomer TIBEMA, sulfonated polyvinyl alcohol, citric acid, etc., were directly mixed and crosslinked using the W / W emulsion method of Example 3, so that iodine was uniformly distributed in the microsphere network. To achieve a development effect similar to that of Example 1, 6.0 g of TIBEMA was added.

[0048] Experimental Example 1 1. Particle size and morphology detection Methods: The wet particle size distribution of each sample was measured using a laser particle size analyzer (Malvin Mastersizer 3000). The particle size and distribution width were expressed as the volume average particle size D50 and the distribution coefficient SPAN = (D90-D10) / D50. The surface and cross-sectional morphology of the microspheres were observed using scanning electron microscopy (SEM).

[0049] Table 1. Results of particle size and morphology testing

[0050] Results (see Table 1): The D50 of Examples 1-8 were all within the selected sieve size range, and the SPAN was <0.8, indicating a narrow particle size distribution. SEM observation showed that the products of all examples were spherically regular with smooth surfaces, and the core-shell boundary was clearly visible in the cross-section. In Comparative Example 3 (W / O method), some microspheres were adhered and deformed, with an SPAN of 1.5, indicating poor particle size uniformity. Comparative Example 4 (coreless-shell-free) showed no layered structure in the cross-section and was homogeneous.

[0051] 2. Iodine content determination and CT imaging properties Methods: The iodine content of dry microspheres was determined by oxygen flask combustion-ion chromatography. Each microsphere was suspended in physiological saline at a concentration of 10 mg / mL. The CT value (HU) of each sample suspension was measured using clinical CT (120 kVp, Siemens SOMATOM Definition Flash), with physiological saline serving as a blank control. Standard curves were prepared using iohexol solutions with iodine concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mg / mL.

[0052] Table 2. Results of Iodine Content and CT Visualization Tests

[0053] Results (see Table 2): In Example 1, the iodine content was 95 mg / g, and the HU value was 320 HU higher than the blank, equivalent to a signal of approximately 3.2 mg I / mL iohexol solution. In Example 7, the iodine content was as high as 195 mg / g, approximately 580 HU higher. In Example 6, the iodine content was 55 mg / g, still approximately 180 HU higher, meeting the intraoperative imaging requirements. In Comparative Example 4, to achieve imaging similar to Example 1, the iodine content needed to reach 350 mg / g. The iodine content of Comparative Example 1 was consistent with that of Example 1, and the iodine contents of Comparative Examples 2 and 3 were also basically consistent with those of Example 1 (the core parts were the same), proving that the iodine content was mainly determined by S1.

[0054] Table 3. Results of elasticity and duct performance tests

[0055] 4. Drug loading and in vitro sustained-release assay Methods: 1 mL of wet microspheres was mixed with 1 mL of doxorubicin solution (5 mg / mL, pH 6.5) and magnetically stirred at 30 °C. After 30 min, the mixture was centrifuged and the supernatant was collected. The absorbance was measured by UV-Vis (480 nm) to calculate the loading (based on the difference between the initial and residual amounts). Loading rate = (initial amount - residual amount) / initial amount × 100%. After loading, the microspheres were transferred to 10 mL of PBS (pH 7.4) and a release experiment was conducted at 37 °C. Samples were taken periodically to measure the drug concentration, and a cumulative release curve was plotted.

[0056] Table 4. Drug Loading and Sustained-Release Test Results

[0057] Results (see Table 4): Example 1 had a loading capacity of 58 mg / mL, a loading rate >99%, a cumulative release of 18% over 24 hours, and a release of 75% over 14 days. Example 4 had a loading capacity of 42 mg / mL, which was still clinically significant. Example 5 had a loading capacity of 62 mg / mL. In Example 7, due to the large core and relatively small shell proportion, the loading capacity decreased to 46 mg / mL. Comparative Example 2 had a loading capacity of only 1.8 mg / mL, demonstrating that sulfonic acid group modification is the chemical basis for drug loading function. Comparative Example 4 had a loading capacity of only 18 mg / mL, because the iodine monomer occupied the shell network space, shielding and diluting the sulfonic acid groups. Comparative Examples 1 and 3 were comparable to Example 1, indicating that their shell drug loading function was not affected.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a radiopaque embolic microsphere, characterized in that, The preparation steps include the following: S1. Dissolve 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate and sebacic acid diacrylate in an ethanol / water mixed solvent, add polyvinylpyrrolidone, and add potassium persulfate under inert gas protection to carry out precipitation polymerization reaction. After the reaction is completed, centrifuge, wash, dialyze and dry to obtain degradable developing core seeds. S2. Polyvinyl alcohol is dissolved in water, activated with an alkaline substance, reacted with 1,3-propanesulfonate lactone, neutralized, dialyzed, and dried to obtain polyvinyl alcohol modified with sulfonic acid groups. S3. The biodegradable developing core seed, sulfonic acid group-modified polyvinyl alcohol, citric acid, catalyst, and water are mixed and dispersed to obtain a dispersed phase; a polyethylene glycol aqueous solution is used as the continuous phase. The dispersed phase is added to the continuous phase under stirring to form a water-in-water emulsion; then, heating is used to cause the polyvinyl alcohol modified with sulfonic acid groups inside the dispersed phase droplets to undergo esterification, cross-linking and curing with citric acid to obtain crude core-shell microspheres. S4. The crude core-shell microspheres are diluted, demulsified, washed, and sieved to obtain the biodegradable and developable core-shell embolic microspheres.

2. The preparation method according to claim 1, characterized in that, The mass ratio of 2-(2,3,5-triiodobenzoyloxy)ethyl methacrylate, sebacic acid diacrylate, ethanol / water mixed solvent, polyvinylpyrrolidone, and potassium persulfate in S1 is 1:(0.2-0.5):100:(0.3-2):(0.01-0.03).

3. The preparation method according to claim 1, characterized in that, The precipitation polymerization reaction in S1 is carried out at a temperature of 60-75℃ for 6-10 hours.

4. The preparation method according to claim 1, characterized in that, In the S1 ethanol / water mixed solvent, the volume ratio of ethanol to water is 1:(0.8-1.5).

5. The preparation method according to claim 1, characterized in that, The mass ratio of polyvinyl alcohol to 1,3-propanesulfonic acid lactone in S2 is 1:(0.3-0.6).

6. The preparation method according to claim 1, characterized in that, The reaction temperature in S2 is 20-40℃, and the reaction time is 8-16 hours.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the biodegradable developing core seed, sulfonic acid group-modified polyvinyl alcohol, citric acid, catalyst, and water in S3 is (10-40):100:(15-35):(1.5-2.5):(800-1000):.

8. The preparation method according to claim 1, characterized in that, The mass concentration of the polyethylene glycol aqueous solution in S3 is 20-40%, and the molecular weight of the polyethylene glycol is 8000-35000.

9. The preparation method according to claim 1, characterized in that, The volume ratio of the dispersed phase to the continuous phase in S3 is 1:(5-20).

10. Imprintable embolic microspheres prepared by the preparation method according to any one of claims 1-9.

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