Polyvinyl alcohol embolization microsphere with photodynamic effect and preparation method thereof

Abstract

The invention discloses a polyvinyl alcohol (PVA) embolization microsphere with a photodynamic effect. The preparation method comprises the following steps: modifying polyvinyl alcohol by adopting an olefinic unsaturated carboxylic acid monomer to obtain modified polyvinyl alcohol, carrying out copolymerization reaction on the modified polyvinyl alcohol and a cross-linking agent free radical to form carboxyl functionalized PVA microspheres, and covalently bonding a photosensitizer to the surfaces of the PVA microspheres by adopting 1, 1 '-carbonyldiimidazole CDI-mediated esterification reaction; the preparation method comprises the following steps: modifying polyvinyl alcohol by adopting an olefinic unsaturated carboxylic acid monomer, preparing carboxylated PVA microspheres with adjustable particle size (100-800 microns) through W / O emulsion crosslinking free radical copolymerization, and then coupling with a CDI activated photosensitizer under the condition that the pH is 5.0-6.5; the obtained microspheres are crosslinked with a photosensitizer through chemical bonds, and active oxygen (ROS) such as singlet oxygen (O) can be efficiently generated under laser irradiation; the sphericity of the microspheres is complete, and the swelling ratio is controllable.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical materials and interventional medicine, and specifically to a preparation method and application of photosensitizer-functionalized polyvinyl alcohol embolization microspheres. Background Art

[0002] Transarterial Chemoembolization (TACE) remains the first-line interventional treatment option for patients with intermediate and advanced liver cancer. However, the following clinical pain points exist: (1) Systemic toxicity caused by chemotherapeutic drugs: The standard TACE protocol uses an embolizing agent-chemotherapeutic drug combined embolization microsphere, but the released chemotherapeutic drugs can cause grade III-IV myelosuppression (platelets < 50×10 9 / L) and liver function damage (the Child-Pugh score decreases by ≥2 points in about 20% of patients after surgery) in 30-50% of patients.

[0003] (2) Limitations in efficacy: Clinical data show that the objective remission rate after TACE treatment is only 35-60%, and the median survival period is less than 20 months.

[0004] Therefore, there is an urgent need to develop new embolizing agents to replace traditional chemotherapeutic drug-loaded embolizing agents. Photodynamic Therapy (PDT) uses a laser to excite a photosensitizer to generate singlet oxygen (¹O 2 ) at a specific wavelength, which oxidizes intracellular biomacromolecules and induces cell death. Compared with chemotherapy, PDT has the advantages of strong spatio-temporal controllability, low systemic toxicity, and no drug resistance. Developing a new photodynamic embolization microsphere carrier system that can be delivered by intervention is of great significance.

[0005] Recently, some studies have attempted to combine photodynamic therapy with interventional embolization therapy for tumor treatment. For example, Liu et al. (Biomaterials 2023, 296, 122094) developed an acid-responsive calcium phosphate nanodrug delivery system, which loaded Ce6 and thrombin through a one-pot method to construct a multi-pathway tumor treatment platform. In the acidic tumor microenvironment, the nanoparticles rapidly dissociated to release thrombin, triggering the conversion of fibrinogen to fibrin, and then blocking blood vessels. At the same time, the photodynamic effect mediated by the photosensitizer Ce6 consumed oxygen and damaged blood vessels, synergistically enhancing the therapeutic effect. However, this method has the following problems: 1. Carrier instability: At blood pH (7.35-7.45), the solubility of calcium phosphate reaches 12.8 mg / mL, resulting in premature release of the photosensitizer at non-target sites. 2. Coagulation risk: The released thrombin increases the incidence of venous thrombosis. 3. Embolization irreversibility: The degradation of calcium phosphate leads to an embolization duration of <14 days and a high blood vessel recanalization rate.

[0006] Therefore, the present invention uses PVA embolization microspheres that will not be decomposed and cause permanent embolization, combines a photosensitizer with low toxicity and side effects and can be repeatedly activated with the embolization microspheres, and is expected to develop a new type of photodynamic embolization microsphere carrier system. Summary of the Invention

[0007] Aiming at the clinical bottlenecks such as the systemic toxicity and drug resistance of chemotherapeutic drugs in traditional TACE therapy, the present invention proposes a chemotherapy-independent synergistic treatment strategy and constructs a photosensitizer-functionalized polyvinyl alcohol (PVA) embolization microsphere system.

[0008] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a polyvinyl alcohol embolization microsphere with a photodynamic effect. Polyvinyl alcohol is modified with an ethylenically unsaturated carboxylic acid monomer to obtain modified polyvinyl alcohol. The modified polyvinyl alcohol undergoes a free radical copolymerization reaction with a crosslinking agent to form a carboxyl-functionalized polyvinyl alcohol microsphere. A photosensitizer is covalently fixed on the surface of the carboxyl-functionalized polyvinyl alcohol microsphere through an esterification reaction to obtain a polyvinyl alcohol embolization microsphere with a photodynamic effect; wherein the polyvinyl alcohol after vinyl modification contains polymerizable double bonds, and the ethylenically unsaturated carboxylic acid monomer is one or more of maleic anhydride, methacrylic acid, and methacrylic acid derivatives.

[0009] Further, the free radical copolymerization reaction is carried out under an ammonium persulfate and sodium bisulfite initiation system.

[0010] Further, the photosensitizer is one or more of benzothiadiazine compounds, porphyrin compounds, and their derivatives; the derivatives contain carboxyl functional groups that can undergo an esterification reaction with the hydroxyl groups of PVA.

[0011] The present invention also provides a preparation method of the above-mentioned polyvinyl alcohol embolization microsphere with a photodynamic effect, including the following steps: (1) Dissolve polyvinyl alcohol in a buffer solution with a pH of 4-6, add an ethylenically unsaturated carboxylic acid monomer, and react at 50-60 °C to form a vinyl-modified prepolymer solution; (2) Mix the vinyl-modified prepolymer solution with an aqueous phase containing a surfactant and then inject it into a liquid paraffin oil phase, and stir at a rotation speed of 400-600 rpm to form a W / O emulsion; (3) Add a crosslinking agent to the W / O emulsion, and under a free radical initiation system, continuously stir at 45-60 °C and 300-600 rpm for 4-8 h to complete free radical copolymerization, and obtain carboxyl-functionalized polyvinyl alcohol microspheres after washing with ethanol and vacuum drying; (4) React the carboxyl-functionalized polyvinyl alcohol microspheres with a photosensitizer under the conditions of pH 5.0 - 6.5 for 18 - 36 hours to obtain photodynamic embolization microspheres. The photosensitizer is a photosensitizer carboxylic acid derivative activated by 1,1'-carbonyldiimidazole (CDI).

[0012] Further, in step (1), the molar ratio of the ethylenically unsaturated carboxylic acid monomer to polyvinyl alcohol is 1:0.5 - 1.2.

[0013] Further, in step (1), the buffer solution contains C3 - C6 hydroxycarboxylic acid; preferably, the buffer solution is an aqueous solution of one or more of acetic acid - sodium acetate, lactic acid, malic acid, tartaric acid, and citric acid, and the mass concentration of the buffer solution is 20 - 40%; the mass concentration of polyvinyl alcohol in the vinylated modified prepolymer solution is 15 - 40%.

[0014] Further, in step (2), the aqueous phase containing a surfactant, and the concentration of the surfactant in this aqueous phase is 1 - 3 wt%; the surfactant is one or more of alkylbenzene sulfonates, α-olefin sulfonates, and sulfosuccinates, and the addition amount of the surfactant is 3 - 10 wt% of the mass of polyvinyl alcohol in step (1).

[0015] Further, in step (2), the volume ratio of the liquid paraffin oil phase to the aqueous phase is 4:1.

[0016] Further, in step (3), the free radical initiation system is a water-soluble oxidation - reduction agent, comprising a combination of ammonium persulfate and sodium bisulfite, and the molar ratio of ammonium persulfate to sodium bisulfite is 1:1.2 - 1.5. The cross-linking agent is a bisacrylamide derivative, and the mass ratio of the cross-linking agent to the W / O emulsion is 0.5 - 2 wt%; the total mass ratio of ammonium persulfate and sodium bisulfite to the W / O emulsion is 1 - 2.5 wt%.

[0017] The present invention also provides the application of the above-mentioned polyvinyl alcohol embolization microspheres with photodynamic effect in the preparation of interventional embolization products.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The present invention constructs PVA microspheres with both a cross-linked skeleton (vinyl group) and active carboxyl groups through free radical copolymerization of vinylated modified PVA and ethylenically unsaturated carboxylic acid monomers.

[0019] The functionalization of traditional PVA microspheres relies on a single chemical modification (such as hydroxyl activation), while the present invention directly introduces carboxyl groups through copolymerization, significantly improving the efficiency and stability of subsequent photosensitizer loading. The loading of traditional photosensitizers mostly relies on physical adsorption or non-specific binding. The present invention realizes the efficient and directional fixation of photosensitizers through chemical bonding, avoiding the leakage risk in in vivo applications.

[0020] The present invention provides a preparation method of photodynamic PVA embolization microspheres, with a simple synthesis method, which is conducive to large-scale production; The present invention has low requirements for the equipment used, and the reagents used are all common reagents, which are environmentally friendly, non-toxic and harmless; The photodynamic embolization microspheres synthesized by the present invention based on PVA materials have complete morphology, uniform size, good dispersibility and good biocompatibility, showing great application potential in the field of interventional medicine; The embolization microspheres have the ability to release ROS reactive oxygen species, and this property enables them to release ROS reactive oxygen species to efficiently kill tumor cells. Description of the Drawings

[0021] Figure 1 Optical microscope photograph (5X) of the PVA embolization microspheres prepared in Example 1 of the present invention; Figure 2 Optical microscope photograph (scale bar is 500μm) of the photodynamic PVA embolization microspheres prepared in Example 1 of the present invention; Figure 3 Water absorption rate pictures of PVA embolization microspheres and PVA photodynamic embolization microspheres based on PVA materials prepared in Example 1 of the present invention; Figure 4 Fourier transform infrared spectrum of the PVA photodynamic embolization microspheres based on PVA materials prepared in Example 1 of the present invention; Figure 5 Embolization image of the PVA photodynamic embolization microspheres based on PVA materials prepared in Example 2 of the present invention in a simulated catheter; Figure 6 Embolization image of the PVA photodynamic embolization microspheres based on PVA materials prepared in Example 2 of the present invention in a simulated thinner catheter; Figure 7 Scanning electron microscope picture of the freeze-dried PVA photodynamic embolization microspheres based on PVA materials prepared in Example 2 of the present invention; Figure 8 Ultraviolet-visible light photometer image of the PVA photodynamic embolization microspheres based on PVA materials prepared in Example 2 of the present invention; Figure 9 Evaluation of the ability of PVA photodynamic embolization microspheres loaded with photosensitizer Ce6 and its control group prepared in Example 3 of the present invention to generate ROS under laser irradiation; Figure 10Fluorescence confocal images of the cytotoxicity of Hepg2 liver cancer cells under laser irradiation of a PVA photodynamic embolization microsphere loaded with photosensitizer Ce6 constructed from a PVA material prepared in Example 3 of the present invention and its control group; Figure 11 Cell survival images of the cytotoxicity of Hepg2 liver cancer cells under laser irradiation of a PVA photodynamic embolization microsphere loaded with photosensitizer Ce6 constructed from a PVA material prepared in Example 3 of the present invention and its control group. Detailed implementation manners

[0022] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] A preparation method of a photodynamic PVA embolization microsphere, and the specific steps are as follows: Step 1: Preparation of vinylated PVA prepolymer: (1) Dissolve polyvinyl alcohol (Maclean, Mw 31000 - 50000, 98 - 99% hydrolysis) in a pH 5.5 acetic acid - sodium acetate buffer solution (purchased from Yuanye), control the mass concentration of the solution to be 20 ± 2%, and dissolve it under stirring at 50°C and 300 rpm for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2), and react at 60°C for 2 hours to form a vinylated modified prepolymer solution; (3) Cool the vinylated modified prepolymer solution to 25°C, wash it 3 times with 75% ethanol, vacuum dry it at 40°C, and then redissolve it in deionized water to obtain a PVA prepolymer solution (solid content 18 ± 1%).

[0024] Step 2: Microsphere cross - linking and forming: (1) Mix the PVA prepolymer solution with Span80 (Energy Chemical, 3 wt%) and inject it into liquid paraffin oil (Sigma - Aldrich, water - oil ratio 1:4); (2) Form a W / O emulsion under stirring at 500 rpm, and add N,N'-methylenebisacrylamide (MBA, 0.8 wt%); (3) Cross - link in a 55°C water bath for 6 hours, wash it 3 times with petroleum ether and anhydrous ethanol in sequence, and freeze - dry to obtain carboxyl - functionalized polyvinyl alcohol microspheres.

[0025] Step 3: Photosensitizer coupling: (1) The carboxylated photosensitizer Ce6-COOH (Xi'an Qiyue Biotechnology) and CDI (MREDA, molar ratio 1:1.2) were dissolved in pH 6.0 MES buffer (Xi'an Reagents) and activated at 25°C in the dark for 30 minutes to obtain an activated solution; (2) adding the carboxyl functionalized polyvinyl alcohol microspheres prepared in step 2 to the activation solution, maintaining the pH at 5.5-6.0, and reacting at 25° C. with shaking for 24 hours; (3) After the reaction, the microspheres were washed three times with 0.1 M PBS to remove the uncoupled photosensitizer and then freeze-dried to obtain photodynamic embolization microspheres.

[0026] Principle process: Functional modification of PVA molecular chain The double bond functionalization of the polyvinyl alcohol (PVA) molecular chain is carried out by methacrylic acid or maleic anhydride, and a polymerizable double bond (-CH=CH-COOH) is introduced into the PVA main chain. The grafted carboxyl group provides a reaction site for the subsequent photosensitizer coupling.

[0027] The specific steps of maleic anhydride to modify the double bond functionalization of polyvinyl alcohol molecular chain are as follows: 1. Raw material pretreatment: Polyvinyl alcohol (McLean, Mw 31000-50000, 98-99% hydrolyzed) was dissolved in pH 5.5 acetic acid-sodium acetate buffer (purchased from source leaves) (concentration 5-10% w / v), heated to 80-90°C and stirred continuously until completely dissolved to obtain a PVA aqueous solution.

[0028] Maleic anhydride activation: Dissolve maleic anhydride (MAH) in N,N-dimethylformamide (McLean, 99% biotech grade) (concentration 10-20% w / v) and set aside.

[0029] 2. Esterification reaction (double bond introduction) Slowly drop the maleic anhydride solution into the PVA aqueous solution, maintaining a molar ratio of PVA: maleic anhydride = 1:0.5~1:2. Add the catalyst p-toluenesulfonic acid (PTSA, p-toluenesulfonic acid: PVA = 0.5-2% w / w), and adjust the pH to 2-3. Under nitrogen protection, react at 60-80°C for 4-8 hours, and continue stirring (300-500 rpm).

[0030] Microsphere cross-linking and curing ①W / O emulsion template construction: The prepolymer solution is mixed with an aqueous phase containing a surfactant (such as Span 80), injected into a liquid paraffin oil phase, and formed into water-in-oil (W / O) emulsion droplets by high-speed stirring (400-600 rpm).

[0031] ②Cross-linking reaction: Add a crosslinking agent (such as N,N'-methylenebisacrylamide) to the emulsion and continuously stir at 55 ± 2 °C for 4 - 6 hours to complete free radical copolymerization crosslinking.

[0032] Functional modification of photosensitizer Through the CDI-mediated carboxylic acid activation mechanism, covalently modify the carboxylated photosensitizer (Ce6-COOH) onto the PVA molecule. Example 1

[0033] Step 1: Preparation of vinylated PVA prepolymer (1) Dissolve 1 g of PVA in 4 g of acetic acid-sodium acetate buffer solution with pH 5.0, control the mass concentration of PVA in the solution to be 20 ± 2%, and dissolve it at 50 °C under stirring at 300 rpm for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2), and react at 60 °C for 2 hours to form a vinylated solution; (3) Cool the reaction solution to 25 °C, wash it 3 times with 75% ethanol, dry it in vacuum at 40 °C and then redissolve it in deionized water to obtain a vinylated PVA prepolymer solution (solid content 18 ± 1%).

[0034] Step 2: Crosslinking and forming of microspheres: (1) Mix the vinylated PVA prepolymer solution with an aqueous phase containing 0.1 g of Span80 (accounting for 3 wt% of the aqueous phase), and inject it into liquid paraffin oil (water-oil ratio 1:4); (2) Form a W / O emulsion under stirring at 500 rpm, and add N,N'-methylenebisacrylamide (MBA, accounting for 0.8 wt% in the W / O emulsion); (3) Crosslink in a water bath at 55 °C for 6 hours, wash it 3 times with petroleum ether and absolute ethanol in sequence, and freeze-dry to obtain carboxyl-functionalized PVA embolization microspheres.

[0035] Step 3: Modification of photosensitizer on the surface of microspheres (1) Dissolve the carboxylated photosensitizer Ce6-COOH (20 mg) and CDI (molar ratio 1:1.2) in 5 ml of MES buffer solution with pH 6.0, and activate it in the dark at 25 °C for 30 minutes; (2) Add 1 g of the carboxyl-functionalized PVA embolization microspheres in Step 2 to the mixture in (1) of Step 3, maintain the pH at 5.5 - 6.0, and react with shaking at 25 °C for 24 hours; (3) After the reaction, wash it 3 times with 0.1 M PBS to remove the uncoupled photosensitizer, and freeze-dry to obtain photodynamic embolization microspheres. Example 2

[0036] Step 1: Preparation of vinylated PVA prepolymer (1) Dissolve 1 g of PVA in 4.5 g of acetic acid - sodium acetate buffer solution with pH 4.0 (control the PVA concentration at 18%), and dissolve it at 50 °C and 300 rpm for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2), and react at 50 °C for 2 hours to form an alkenylated solution; (3) Cool to 25 °C, wash 3 times with 75% ethanol, vacuum dry at 40 °C and then redissolve to obtain a prepolymer solution with a solid content of 17%.

[0037] Step 2: Microsphere cross - linking and forming (1) Mix the alkenylated PVA prepolymer solution with an aqueous phase containing 0.1 g of Span80 (accounting for 3 wt% of the aqueous phase), and inject it into liquid paraffin oil (water - oil ratio 1:4); (2) Form a W / O emulsion under stirring at 500 rpm, and add N,N'-methylenebisacrylamide (MBA, accounting for 0.8 wt% in the W / O emulsion); (3) Stir at 45 °C and 300 rpm for 4 hours, wash with petroleum ether / ethanol, and freeze - dry to obtain carboxylated PVA microspheres.

[0038] Step 3: Surface modification with photosensitizer (1) Activate Ce6 - COOH and CDI (1:1.2) in MES buffer solution with pH 5.0 for 30 minutes; (2) Add 1 g of the carboxyl - functionalized PVA embolization microspheres in Step 2 to 4 g of the mixture in (1) of Step 3, and oscillate at pH 5.0 and 25 °C for 18 hours; (3) After the reaction, wash 3 times with 0.1 M PBS to remove the uncoupled photosensitizer, and freeze - dry to obtain the photodynamic embolization microspheres.

[0039] This experimental example is the minimum parameter combination. As the pH and reaction temperature in Step 1, the emulsification rotation speed, cross - linking time, and cross - linking temperature in Step 2 are decreased, the size of the microspheres becomes larger, ranging from 500 - 800 μm. Example 3

[0040] Step 1: Preparation of alkenylated PVA prepolymer (1) Dissolve 1 g of PVA in 3.8 g of buffer solution with pH 6.0 (PVA concentration 22%), and dissolve it at 60 °C for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2) and react at 60 °C for 2 hours to form an alkenylated solution; (3) Cool the reaction solution to 25 °C, wash 3 times with 75% ethanol, vacuum dry at 40 °C and then redissolve in deionized water to obtain an alkenylated PVA prepolymer solution (solid content 19 ± 1%) Step 2: Microsphere crosslinking and forming (1) Mix the vinylated PVA prepolymer solution with the aqueous phase containing 0.1 g Span80 (3 wt% of the aqueous phase), and inject it into liquid paraffin oil (water-oil ratio 1:4); (2) Form a W / O emulsion under stirring at 600 rpm, and add N,N'-methylenebisacrylamide (MBA, 0.8 wt% in the W / O emulsion); (3) Stir at 60 °C and 600 rpm for 8 hours, and perform post-treatment to obtain microspheres.

[0041] Step 3: Surface modification with photosensitizer (1) Activate Ce6-COOH and CDI (1:1.2) in a pH 5.0 MES buffer for 30 minutes; (2) Add 1 g of the carboxyl-functionalized PVA embolization microspheres from Step 2 to 4 g of the mixture in (1) of Step 3, and oscillate at pH 6.5 and 25 °C for 24 hours; (3) After the reaction, wash with 0.1 M PBS three times to remove the uncoupled photosensitizer, and freeze-dry to obtain the photodynamic embolization microspheres.

[0042] This experimental example is the maximum parameter combination. As the pH and reaction temperature in Step 1, the emulsification rotation speed, crosslinking time, and crosslinking temperature in Step 2 are increased, the size of the microspheres becomes smaller, being 100 - 200 μm, and the mechanical strength of the microspheres is 1.5 times greater than that in Example 1. Example 4

[0043] Step 1: Preparation of vinylated PVA prepolymer (1) Dissolve 1 g of PVA in 4 g of pH 5.0 acetic acid-sodium acetate buffer, control the mass concentration of PVA in the solution to be 20 ± 2%, and dissolve it under stirring at 50 °C and 300 rpm for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2), and react at 60 °C for 2 hours to form a vinylated solution; (3) Cool the reaction solution to 25 °C, wash it three times with 75% ethanol, vacuum-dry at 40 °C, and then redissolve it in deionized water to obtain a vinylated PVA prepolymer solution (solid content 18 ± 1%).

[0044] Step 2: Microsphere crosslinking and forming: (1) Mix the vinylated PVA prepolymer solution with the aqueous phase containing 0.1 g Span80 (3 wt% of the aqueous phase), and inject it into liquid paraffin oil (water-oil ratio 1:4); (2) A W / O emulsion was formed under stirring at 500 rpm, and N,N'-methylenebisacrylamide (MBA, 0.8 wt% in the W / O emulsion) was added. (3) Crosslinking was carried out in a water bath at 55 °C for 3 hours. It was washed three times with petroleum ether and absolute ethanol in sequence, and then freeze-dried to obtain carboxyl-functionalized PVA embolization microspheres.

[0045] Step 3: Modification of photosensitizer on the surface of microspheres (1) Carboxylated photosensitizer Ce6-COOH (20 mg) and CDI (molar ratio 1:1.2) were dissolved in 5 ml of pH 6.0 MES buffer solution, and activated in the dark at 25 °C for 30 minutes. (2) 1 g of the carboxyl-functionalized PVA embolization microspheres obtained in Step 2 was added to 4 g of the mixture in (1) of Step 3, and the pH was maintained at 5.5 - 6.0. The reaction was carried out with shaking at 25 °C for 12 hours. (3) After the reaction, it was washed three times with 0.1 M PBS to remove the uncoupled photosensitizer, and then freeze-dried to obtain photodynamic embolization microspheres.

[0046] In this example, photodynamic embolization microspheres loaded with photosensitizer Ce6 were prepared, but the reaction time was shortened by half. The size of the microspheres in this example was not as uniform as that in Example 1, being 100 - 800 μm. And shortening the crosslinking time by half led to a 60% reduction in the yield of the microspheres compared with Example 1. Example 5

[0047] Step 1: Preparation of vinylated PVA prepolymer (1) 1 g of PVA was dissolved in 4 g of pH 5.0 acetic acid - sodium acetate buffer solution, and the mass concentration of PVA in the solution was controlled to be 20 ± 2%. It was dissolved under stirring at 50 °C and 300 rpm for 120 minutes. (2) Methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2) was added, and the reaction was carried out at 60 °C for 2 hours to form a vinylated solution. (3) The reaction solution was cooled to 25 °C, washed three times with 75% ethanol, vacuum-dried at 40 °C and then redissolved in deionized water to obtain a vinylated PVA prepolymer solution (solid content 18 ± 1%).

[0048] Step 2: Crosslinking and molding of microspheres: (1) The photodynamic embolization microsphere solution was mixed with an aqueous phase containing 0.12 g of Span80 (3.5 wt% in the aqueous phase), and injected into liquid paraffin oil (water - oil ratio 1:4). (2) A W / O emulsion was formed under stirring at 550 rpm, and N,N'-methylenebisacrylamide (MBA, 0.8 wt% in the W / O emulsion) was added. (3) Crosslink for 6 hours in a 55 °C water bath, wash three times successively with petroleum ether and absolute ethanol, and freeze-dry to obtain carboxyl-functionalized PVA embolization microspheres.

[0049] Step 3: Modify the surface of the microspheres with a photosensitizer (1) Dissolve carboxylated photosensitizer Ce6-COOH (20 mg) and CDI (molar ratio 1:1.2) in 5 ml of pH 6.0 MES buffer solution, and activate in the dark at 25 °C for 60 minutes; (2) Add 1 g of the carboxyl-functionalized PVA embolization microspheres in Step 2 to 4 g of the mixture in (1) of Step 3, maintain the pH at 5.5 - 6.0, and react with shaking at 25 °C for 36 hours; (3) After the reaction, wash three times with 0.1 M PBS to remove the uncoupled photosensitizer, and freeze-dry to obtain the photodynamic embolization microspheres.

[0050] In this example, photodynamic embolization microspheres loaded with photosensitizer Ce6 were prepared. However, in step (2), the activation time was increased from 30 minutes to 60 minutes, and in step (3), the reaction time between the photosensitizer and the PVA microspheres was increased from 24 h to 36 h. By detecting the absorbance of the supernatant before and after the reaction through UV, it was verified that the content of the photosensitizer in the photodynamic embolization microspheres of this example was 1.5 times that in Example 1. Example 6

[0051] Step 1: Preparation of vinylated PVA prepolymer (1) Dissolve 1 g of PVA in 4 g of pH 5.0 acetic acid - sodium acetate buffer solution, control the mass concentration of PVA in the solution to be 20 ± 2%, and dissolve under stirring at 50 °C and 300 rpm for 120 minutes; (2) Add methacrylic acid (molar ratio of PVA:methacrylic acid = 1:1.2), and react at 60 °C for 2 hours to form a vinylated PVA prepolymer solution; (3) Cool the reaction solution to 25 °C, wash three times with 75% ethanol, vacuum dry at 40 °C, and then redissolve in deionized water to obtain a vinylated PVA prepolymer solution (solid content 18 ± 1%).

[0052] Step 2: Crosslinking and forming of microspheres: (1) Mix the photodynamic embolization microsphere solution with an aqueous phase containing 0.1 g of Span80 (3 wt% of the aqueous phase), and inject it into liquid paraffin oil (water - oil ratio 1:4); (2) Form a W / O emulsion under stirring at 800 rpm, and add N,N'-methylenebisacrylamide (MBA, 0.8 wt% in the W / O emulsion); (3) Crosslink in a 55 °C water bath for 6 hours. Introduce 0.5 wt% PVA in the emulsion as a stabilizer. Wash three times with petroleum ether and absolute ethanol in sequence, and then freeze-dry to obtain carboxyl-functionalized PVA embolization microspheres.

[0053] Step 3: Modify the photosensitizer on the surface of the microspheres (1) Dissolve carboxylated photosensitizer Ce6-COOH (20 mg) and CDI (molar ratio 1:1.2) in 5 ml of pH 6.0 MES buffer solution, and activate in the dark at 25 °C for 30 minutes; (2) Add 1 g of the embolization microspheres in Step 2 to 4 g of the mixture in (1) of Step 3, maintain the pH at 5.5 - 6.0, and react with shaking at 25 °C for 24 hours; (3) After the reaction, wash three times with 0.1 M PBS to remove the uncoupled photosensitizer, and then freeze-dry to obtain the photodynamic embolization microspheres.

[0054] In this example, photodynamic embolization microspheres loaded with photosensitizer Ce6 were prepared. However, the emulsification speed was increased and a stabilizer was added to control the particle size of the microspheres. The size of the microspheres in this example was smaller than that in Example 1, being 100 - 300 μm. However, there was an adhesion phenomenon among these microspheres. Verification example

[0055] (1) Observe and photograph the carboxyl-functionalized PVA embolization microspheres prepared in Example 1 under a microscope. The results are as Figure 1 shown. The carboxyl-functionalized PVA embolization microspheres have a good spherical structure and uniform size. The specific size is 200 - 400 μm (the scale bar is 500 μm).

[0056] (2) Observe and photograph the photodynamic embolization microspheres prepared in Example 1 under a microscope. The results are as Figure 1 shown. Loading the photosensitizer did not destroy the good spherical structure of the embolization microspheres (the scale bar is 500 μm).

[0057] (3) Place 100 mg of the freeze-dried photodynamic embolization microspheres prepared in Example 1 into a 50 ml centrifuge tube, add 30 ml of PBS solution, let it stand for 2 h to allow the microspheres to fully absorb water, centrifuge, aspirate the excess liquid, and weigh after absorbing the unabsorbed PBS solution with filter paper. The results are as Figure 3 shown. The swelling ratio of the PVA embolization microspheres is about 380%, and the photosensitizer does not affect the swelling ratio of the microspheres.

[0058] (4) The infrared image of the photodynamic embolization microspheres prepared in Example 1 is as Figure 4 shown. There is an ester bond absorption peak at 1735 for the photodynamic embolization microspheres, indicating that the photosensitizer was successfully linked to PVA.

[0059] (5)Figure 5 The embolization image of a PVA photodynamic embolization microsphere constructed based on PVA material prepared in Example 2 of the present invention in a simulated catheter is shown. The results indicate that the photodynamic embolization microsphere can completely embolize the entire catheter without leaving gaps, demonstrating that the embolization microsphere has good embolization effect.

[0060] (6) Figure 6 The embolization image of a PVA photodynamic embolization microsphere constructed based on PVA material prepared in Example 2 of the present invention in a simulated thinner catheter is shown. The results indicate that the photodynamic embolization microsphere in the present invention can pass through the thin catheter and has good deformation effect.

[0061] (7) Figure 7 The electron scanning microscope picture of a PVA photodynamic embolization microsphere constructed based on PVA material prepared in Example 2 of the present invention after freeze-drying is shown. The results indicate that the photodynamic embolization microsphere still has good morphology after freeze-drying and its surface is smooth, which helps the microsphere pass through the catheter.

[0062] (8) Figure 8 The ultraviolet-visible light photometer image of a PVA photodynamic embolization microsphere constructed based on PVA material prepared in Example 3 of the present invention is shown. The results indicate that the photodynamic embolization microsphere has an ultraviolet absorption peak of photosensitizer Ce6 at 660 nm, demonstrating the successful loading of the photosensitizer.

[0063] (9) Figure 9 The fluorescence confocal image of the ability to generate ROS of a PVA photodynamic embolization microsphere constructed based on PVA material loaded with photosensitizer Ce6 prepared in Example 3 of the present invention and its control group under laser irradiation is shown. The results indicate that the photodynamic embolization microsphere has a ROS reactive oxygen generation effect close to that of photosensitizer Ce6, demonstrating that the photodynamic embolization microsphere has good reactive oxygen generation effect and the generated ROS reactive oxygen can enter tumor cells.

[0064] (10) Figure 10 The fluorescence confocal picture of the cytotoxicity of a PVA photodynamic embolization microsphere constructed based on PVA material loaded with photosensitizer Ce6 prepared in Example 3 of the present invention and its control group under laser irradiation is shown. The liver cancer cells were seeded in a 96-well plate at a density of 1×10 4 cells per well. After overnight culture, the cells were incubated with the photodynamic embolization microsphere solution for 24 hours, and then irradiated with a 660 nm laser (1 W / cm 2 2) for 5 min in each well, and then stained with AM / PI solution and photographed using fluorescence confocal microscopy. The results indicate that after laser irradiation, most of the cells co-incubated with the photodynamic embolization microsphere group showed red fluorescence, demonstrating that the photodynamic embolization microsphere has good cell killing effect under laser irradiation.

[0065] (11) Figure 11 This is the cell survival image of the cytotoxicity of Hepg2 liver cancer cells under laser irradiation of a PVA photodynamic embolization microsphere loaded with photosensitizer Ce6 constructed from a PVA material prepared in Example 4 of the present invention and its control group. The liver cancer cells were seeded in a 96-well plate at a density of 1×10 4 cells per well. After overnight incubation, the cells were incubated with the photodynamic embolization microsphere solution for 24 hours, and then irradiated with a 660 nm laser (1 W / cm 2 ) for 5 min to the cells in each well. After overnight incubation, the cell viability was evaluated using the MTT method. The results showed that before laser irradiation, the cell survival rate of the photodynamic embolization microsphere group was above 95%, indicating that the photodynamic embolization microspheres had good biocompatibility. After laser irradiation, the cell survival rate of the photodynamic embolization microsphere group was about 20%, indicating that the photodynamic embolization microspheres had good cell killing effect.

[0066] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0067] It should be noted that the above content only illustrates the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements all fall within the protection scope of the claims of the present invention.

Claims

1. A polyvinyl alcohol embolic microsphere with photodynamic effect, characterized in that: Polyvinyl alcohol is modified by using olefinic unsaturated carboxylic acid monomers to obtain modified polyvinyl alcohol, the modified polyvinyl alcohol and a cross-linking agent are subjected to free radical copolymerization to form carboxyl functionalized polyvinyl alcohol microspheres, and a photosensitizer is covalently fixed on the surface of the carboxyl functionalized polyvinyl alcohol microspheres through an esterification reaction to obtain polyvinyl alcohol embolization microspheres with photodynamic effect; wherein the olefinic modified polyvinyl alcohol contains a polymerizable double bond, and the olefinic unsaturated carboxylic acid monomer is one or more of maleic anhydride, methacrylic acid, and methacrylic acid derivatives.

2. The polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 1, characterized in that: The free radical copolymerization reaction is carried out under the initiation system of ammonium persulfate and sodium bisulfite.

3. The polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 1, characterized in that: The photosensitizer is one or more of benzothiadiazine compounds, porphyrin compounds and their derivatives.

4. A method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) dissolving polyvinyl alcohol in a buffer solution of pH 4-6, adding an ethylenically unsaturated carboxylic acid monomer, and reacting at 50-60° C. to form an ethylenically modified prepolymer solution; (2) mixing the olefin-modified prepolymer solution with the aqueous phase containing a surfactant, injecting the mixture into the liquid paraffin oil phase, and stirring at a speed of 400-600 rpm to form a W / O emulsion; (3) adding a crosslinking agent to the W / O emulsion, and continuously stirring at 45-60°C and 300-600 rpm for 4-8 hours under the initiation system of ammonium persulfate and sodium bisulfite to complete free radical copolymerization, and washing with ethanol and vacuum drying to obtain carboxyl functionalized polyvinyl alcohol microspheres; (4) reacting the carboxyl functionalized polyvinyl alcohol microspheres with a photosensitizer at pH 5.0-6.5 for 18-36 hours to prepare photodynamic embolization microspheres.

5. The method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 4, characterized in that: In step (1), the molar ratio of the polyvinyl alcohol to the ethylenically unsaturated carboxylic acid monomer is 1:0.5-1.

2.

6. The method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 4, characterized in that: In step (1), the buffer solution is an aqueous solution of one or more of acetic acid-sodium acetate, lactic acid, malic acid, tartaric acid, and citric acid, and the mass concentration of the buffer solution is 20-40%; the mass concentration of polyvinyl alcohol in the olefin-modified prepolymer solution is 15-40%.

7. The method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 4, characterized in that: In step (2), the mass concentration of the surfactant in the aqueous phase is 1-3%; the surfactant is one or more of alkylbenzene sulfonate, α-olefin sulfonate, and succinate sulfonate, and the added amount of the surfactant is 3-10wt% of the mass of the polyvinyl alcohol in step (1).

8. The method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 4, characterized in that: In step (2), the volume ratio of the liquid paraffin oil phase to the water phase is 4:

1.

9. The method for preparing polyvinyl alcohol embolic microspheres with photodynamic effect according to claim 4, characterized in that: In step (3), the molar ratio of ammonium persulfate to sodium bisulfite is 1:1.2-1.5, the cross-linking agent is a bisacrylamide derivative, the mass ratio of the cross-linking agent to the W / O emulsion is 0.5-2wt%; the mass ratio of the total amount of ammonium persulfate and sodium bisulfite to the W / O emulsion is 1-2.5wt%.

10. Use of the polyvinyl alcohol embolic microspheres with photodynamic effect as claimed in any one of claims 1 to 3 in the preparation of interventional embolic products.

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