Drug sustained-release elastic embolism microspheres, and preparation method and application thereof

Abstract

The application discloses a drug sustained-release elastic embolism microsphere and a preparation method and application thereof, and belongs to the technical field of medical materials. The application is prepared by the following steps: taking an acetic acid aqueous solution in which chitosan is dissolved as a dispersed phase, mixing the dispersed phase and a continuous phase into a W / O monodisperse emulsion template through microfluidic technology, introducing the W / O monodisperse emulsion template into an acceptant phase containing genipin, a W / O surfactant and an organic oil, crosslinking under magnetic stirring for 4-24 hours, post-treating the obtained microspheres to obtain elastic embolism microspheres, and finally dispersing the elastic embolism microspheres in an impregnation liquid in which a hydrophilic antitumor drug is dissolved and performing oscillation treatment, so that the drug sustained-release elastic embolism microsphere is prepared. The application effectively optimizes the drug loading capacity and drug sustained-release performance of the formed microspheres by setting the single chitosan dispersed phase component and adopting the technical strategy of jointly regulating and controlling the microfluidic control emulsification and crosslinking time, so that the effect of simply preparing the drug sustained-release elastic embolism microsphere is achieved.

Description

Technical Field

[0001] This application belongs to the field of medical materials technology, and specifically discloses a drug sustained-release elastic embolization microsphere, its preparation method and application. Background Technology

[0002] Transarterial chemoembolization (TACE) is an important minimally invasive clinical intervention for tumors. Its core principle is to precisely deliver embolic materials into target blood vessels to block blood supply, inducing ischemic necrosis of the lesions. Among these materials, elastic embolic microspheres have become a research hotspot due to their excellent biocompatibility and vascular adaptability. However, traditional embolic microspheres only provide mechanical vascular embolization, resulting in limited clinical efficacy. Furthermore, for patients requiring combined chemotherapy, embolization and chemotherapy must be performed separately, increasing the treatment process and potentially reducing patient tolerance. Therefore, the development of drug-loaded embolic microspheres with sustained-release properties is imperative.

[0003] Patent CN 114712551 B discloses a monodisperse gelatin-chitosan composite embolic microsphere with adjustable degradation performance and elasticity. The process involves treating an aqueous solution of gelatin and chitosan in acetic acid to form an internal phase fluid, then mixing it with an external phase fluid containing a surfactant using microfluidic technology to form a monodisperse water-in-oil emulsion. This emulsion is then mixed with a collection solution containing the crosslinking agent genipin for crosslinking to obtain the monodisperse gelatin-chitosan composite embolic microsphere. This effectively improves the monodispersity of the composite embolic microsphere while giving it controllable adjustable degradation time and elasticity.

[0004] However, the existing technology has obvious shortcomings and defects: First, the molecular chains of the mixed internal phase of chitosan and gelatin are highly entangled, making it difficult to construct a regular drug loading space and drug release network. It does not have a highly efficient drug-carrying carrier structure, making it difficult to achieve synergistic treatment of embolization and chemotherapy. Furthermore, it cannot precisely control the drug release behavior, which is not conducive to the long-term and stable release of drugs and makes it difficult to achieve the ideal clinical prognosis. Summary of the Invention

[0005] This application discloses a drug-releasing elastic embolization microsphere, its preparation method, and its application, effectively solving the technical problem that existing monodisperse gelatin-chitosan composite embolization microspheres do not possess efficient drug loading and sustained-release drug structural characteristics.

[0006] To achieve the above objectives, the technical solution provided in this application is as follows:

[0007] The first aspect of this application provides a method for preparing drug-release elastic embolization microspheres, which includes the following steps:

[0008] Chitosan powder was dissolved in an aqueous acetic acid solution and treated to obtain a clear dispersed phase solution;

[0009] The dispersed phase solution and the continuous phase are mixed using microfluidics to form a W / O monodisperse emulsion template, wherein the continuous phase comprises organic oil and W / O type surfactant;

[0010] Genipin crosslinking agent was dissolved in organic oil containing W / O type surfactant to form an acceptor phase. The W / O monoemulsion template was introduced into the acceptor phase and crosslinked for 4 to 24 hours under magnetic stirring. After the crosslinking reaction was completed, the obtained microspheres were centrifuged, washed and freeze-dried in sequence to obtain elastic embolization microspheres.

[0011] The elastic embolic microspheres are dispersed in an impregnation solution containing a hydrophilic antitumor drug and then shaken in a shaker to obtain drug-release elastic embolic microspheres.

[0012] According to the preferred disclosure of the first aspect, the crosslinking time under magnetic stirring is 4 to 8 hours.

[0013] According to the preferred disclosure of the first aspect, the chitosan has the following characteristics:

[0014] Deacetylation degree >95%; viscosity 50~100 mPa·s;

[0015] Furthermore, the mass-volume ratio of the chitosan to the acetic acid aqueous solution is 1~3% w / v.

[0016] According to the preferred disclosure of the first aspect, the W / O type surfactant is selected from one or more of Span 85 and Span 80;

[0017] And / or, the organic oil is selected from one or more of isopropyl palmitate, soybean oil, liquid paraffin, dimethyl silicone oil and petroleum ether.

[0018] According to the preferred disclosure of the first aspect, the mass-volume ratio concentration of the W / O type surfactant and the organic oil contained in the continuous phase and the receiving phase is 4-6% w / v, and the mass-volume ratio of the genipin crosslinking agent contained in the receiving phase is 2-6 mg / mL.

[0019] According to the preferred disclosure of the first aspect, the temperature of the magnetic stirring is maintained at 35~45°C;

[0020] And / or, the centrifugation speed is set to 2500~3500 rpm, and the centrifugation time is 3~5 minutes;

[0021] And / or, the freeze-drying time is 24 hours;

[0022] And / or, the temperature of the shaker is set to 45°C and the rotation speed is set to 800 rpm.

[0023] According to the preferred disclosure of the first aspect, the hydrophilic antitumor drug is selected from any one of pentafluorouracil, doxorubicin hydrochloride, and tetracycline hydrochloride;

[0024] And / or, the solvent of the impregnation solution contains sodium acetate buffer;

[0025] And / or, the concentration of the hydrophilic antitumor drug in the impregnation solution is 2~8 mg / mL, and the mass-to-volume ratio of the elastic embolization microspheres to the impregnation solution is 4 mg:3 mL.

[0026] According to the preferred disclosure of the first aspect, the microfluidic device of the microfluidic technology includes a quasi-two-dimensional polydimethylsiloxane microfluidic chip, and the channel structure of the polydimethylsiloxane microfluidic chip is a flow focusing structure.

[0027] The second aspect of this application also discloses a drug sustained-release elastic embolization microsphere prepared by the preparation method described in this application, wherein the particle size of the drug sustained-release elastic embolization microsphere is adjustable within the range of 15 to 80 μm and the particle size variation coefficient is less than 5%.

[0028] The third aspect of this application also discloses the application of the drug-release elastic embolization microspheres described herein, specifically, the use of these drug-release elastic embolization microspheres in the preparation of embolic agents. These embolic agents can be used for vascular interventional embolization therapy, and are applicable to conditions including, but not limited to, malignant tumors, benign tumors, vascular malformations, and hemorrhagic diseases. Specifically, they can be used for interventional treatment of conditions such as liver cancer, lung cancer, uterine fibroids, hemangiomas, and ruptured esophageal varices.

[0029] Compared with the prior art, the advantages or beneficial effects of this application include at least:

[0030] The preparation method provided in this application employs a strategy of setting a single chitosan dispersed phase component and using microfluidic control combined with cross-linking time regulation. On one hand, this allows the chitosan molecular chains to undergo orderly cross-linking with a "first unfolding, then cross-linking" process, forming a network structure with regular pore distribution and good connectivity. This provides sufficient and uniform loading sites for drug loading, effectively increasing drug capacity, and also improves the uniformity of drug dispersion within the microspheres, effectively reducing the risk of local burst release. This provides a structural precursor for sustained and stable drug release. On the other hand, the cross-linking density and network porosity of the formed microspheres can be directionally adjusted, effectively optimizing the cross-linked network structure to enable long-term and stable drug release through a slow diffusion mechanism. This increases the local drug concentration at the lesion site to enhance therapeutic efficacy and optimizes the microsphere size performance to precisely match the target vessel size, significantly improving the therapeutic effect of arterial chemoembolization and promoting the development of embolization therapy towards a more precise, efficient, and safer direction. Furthermore, the preparation strategy of this application is easier to scale up and more controllable, providing favorable conditions for subsequent industrialization and clinical application. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 A schematic diagram of the channel structure of the PDMS microfluidic chip provided in this application;

[0033] Figure 2 A schematic diagram of the flow focusing point of the PDMS microfluidic chip channel provided in this application;

[0034] Figure 3 Scanning electron microscope images of the surface of elastic embolization microspheres formed at different crosslinking times provided in this application;

[0035] Figure 4 Scanning electron microscope images of the internal cross-sections of elastic embolization microspheres formed at different cross-linking times provided in this application;

[0036] Figure 5 The swelling properties of the elastic embolic microspheres provided in this application vary with the concentration of the crosslinking agent and the crosslinking time.

[0037] Figure 6 Stress-strain curve of a single elastic embolized microsphere provided in this application;

[0038] Figure 7 The curve showing the change of Young's modulus of the elastic embolization microspheres provided in this application with crosslinking time;

[0039] Figure 8 The drug release kinetics curves of the elastic embolic microspheres provided in this application. Detailed Implementation

[0040] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments described in this application without creative effort are within the scope of protection of this application.

[0041] In the following description of this application, the term "and / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. Here, A and B can be singular or plural; the symbol " / " means "or".

[0042] In the following description of this application, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions mean any combination of such items, including any combination of single or multiple items. For example, "at least one of A, B or C" or "at least one of A, B and C" can mean any one of A, B, and C, or A+B, or A+C, or B+C, or A+B+C, where A, B, and C can be single or multiple.

[0043] In the following description of this application, the order of the sequence numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be specifically determined by its function and internal logic, and does not constitute any limitation on the execution process of this embodiment.

[0044] In the following description of this application, the numerical range should be understood to also specifically disclose each intermediate value between the upper and lower limits of the range. Any intermediate value within a stated range, as well as any other stated value or each smaller range between intermediate values ​​within a stated range, are also included in this embodiment, and the upper and lower limits of the smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, the technical / scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art. While this application describes only preferred materials and methods, similar or equivalent methods and materials may be used in specific embodiments or test cases. All references to this application 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 application shall prevail.

[0046] To address the existing problems, the first aspect of this application provides a method for preparing drug-release elastic embolization microspheres, comprising the following steps S1 to S4:

[0047] S1: Dissolve chitosan powder in an aqueous acetic acid solution and process it into a clear dispersed phase solution;

[0048] S2: The dispersed phase solution and the continuous phase are mixed using microfluidics to form a W / O monodisperse emulsion template, wherein the continuous phase contains organic oil and W / O type surfactant;

[0049] S3: Dissolve the genipin crosslinking agent in an organic oil containing a W / O type surfactant to form an acceptor phase. Introduce the W / O monoemulsion template into the acceptor phase and crosslink under magnetic stirring for 4 to 24 hours. After the crosslinking reaction is completed, centrifuge, wash and freeze-dry the obtained microspheres in sequence to obtain elastic embolization microspheres.

[0050] S4: The elastic embolization microspheres are dispersed in an impregnation solution containing a hydrophilic antitumor drug and then shaken in a shaker to obtain drug-release elastic embolization microspheres.

[0051] This application employs a technique that combines a single chitosan dispersion phase with microfluidic-controlled emulsion and cross-linking time regulation. On one hand, this allows the chitosan molecular chains to undergo orderly cross-linking—a process of "expansion followed by cross-linking"—to form a well-distributed, interconnected network structure with regular pore size. This provides ample and uniform loading sites for drug loading, effectively increasing drug capacity, and also improves the uniformity of drug dispersion within the microspheres, effectively reducing the risk of local burst release. This provides a structural precursor for sustained and stable drug release. On the other hand, the cross-linking density and network porosity of the formed microspheres can be directionally adjusted, effectively optimizing the cross-linked network structure to enable long-term, stable drug release via a slow diffusion mechanism. This increases local drug concentration at the lesion site, enhancing therapeutic efficacy, and optimizes microsphere size performance to precisely match target vessel size, significantly improving the therapeutic effect of arterial chemoembolization and promoting the development of embolization therapy towards greater precision, efficiency, and safety. Furthermore, the preparation strategy of this application is easier to scale up and more controllable, providing favorable conditions for subsequent industrialization and clinical application.

[0052] In possible public examples, the crosslinking time under magnetic stirring described in this application is preferably 4 to 8 hours, thereby effectively adjusting the degree of crosslinking inside the microspheres to optimize drug loading and sustained-release performance. The embodiments of this application use 4, 6, and 8 hours as examples for illustrative description because these three time points precisely cover the lower limit, middle value, and upper limit of the preferred range of this application, comprehensively and clearly presenting the influence of crosslinking time on microsphere performance throughout the preferred range, facilitating the understanding of the core technical logic of this application by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Other time points falling within the range of 4 to 8 hours are all within the scope of protection of this application, and will not be listed individually here.

[0053] In possible disclosed examples, the chitosan described in this application preferably has the following characteristics:

[0054] Deacetylation degree >95%;

[0055] Viscosity is 50~100 mPa·s;

[0056] Furthermore, the mass-volume ratio of the chitosan to the acetic acid aqueous solution is 1~3% w / v.

[0057] It should be noted that this application controls the above-mentioned characteristics of the selected chitosan to ensure sufficient and orderly cross-linking based on the synergistic effect between "dispersion concentration, degree of deacetylation, and viscosity," thereby improving the microsphere network structure and enhancing the uniformity of microsphere molding, while balancing drug loading capacity and sustained-release performance. In this application, a degree of deacetylation of 95%, a viscosity of 80 mPa·s, and a mass-volume ratio concentration of 2% w / v are selected as representative parameters for illustrative description because the chitosan molecular chain extension, dispersion stability, and cross-linking reactivity under this parameter combination are optimal. This combination comprehensively and clearly presents the influence of chitosan characteristics on microsphere performance, facilitating the understanding of the core technical logic of this application by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Other characteristic parameters falling within the range of characteristic parameters are also within the scope of protection of this application, and will not be listed individually here.

[0058] In possible public examples, the W / O type surfactant described in this application is preferably one or more of Span 85 and Span 80. Span 80 is chosen as a representative example in this application because it has relatively high emulsification efficiency, mild dosage, and can ensure uniform microsphere formation and good controllability of particle size distribution, facilitating the understanding of the core technical logic of this application by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Span 85 also falls within the scope of protection of this application, and will not be listed individually here.

[0059] In possible public examples, the organic oil described in this application is preferably one or more of isopropyl palmitate, soybean oil, liquid paraffin, dimethyl silicone oil, and petroleum ether. Isopropyl palmitate is chosen as a representative example in this application for illustrative purposes because it has relatively low volatility and good interfacial stability, which facilitates microsphere molding and subsequent curing and separation, and is convenient for large-scale verification. However, this does not constitute any limitation on the scope of protection of this application. Other organic oils that can achieve the same or similar effects also fall within the scope of protection of this application, and will not be listed individually here.

[0060] In possible disclosed examples, the mass-to-volume ratio concentration of the W / O surfactant to organic oil in both the continuous phase and the receiving phase is 4-6% w / v, and the mass-to-volume ratio of the genipin crosslinking agent in the receiving phase is 2-6 mg / mL. The embodiment of this application uses a mass-to-volume ratio concentration of 4% w / v for the W / O surfactant to organic oil as an example because this concentration is the lower limit of the preferred range of this application. This concentration can stably form a uniform W / O system, ensure regular microsphere formation and uniform particle size, and simultaneously consider subsequent separation and purification and biosafety, which is convenient for those skilled in the art to understand. However, this does not constitute any limitation on the scope of protection of this application. Other mass-to-volume ratios falling within the above range are all within the scope of protection of this application, and will not be listed individually here. Furthermore, the embodiment of the application uses a genipin crosslinking agent... The 2 mg / mL, 4 mg / mL, and 6 mg / mL concentrations are used as examples because these three concentrations precisely cover the lower, middle, and upper limits of the preferred range of this application. They comprehensively and clearly present the influence of the genipin crosslinking agent concentration on the microsphere performance throughout the preferred range, facilitating the understanding of the core technical logic of this application by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Other concentrations falling within the range of 2 to 6 mg / mL are also within the scope of protection of this application, and will not be listed individually here.

[0061] In possible public examples, the magnetic stirring temperature described in this application is preferably 35~45℃, and the rotation speed is 50 rpm, thereby achieving the preparation of microspheres with regular shape, uniform particle size, and stable structure. The embodiment of this application uses a temperature of 40℃ and a rotation speed of 50 rpm for illustrative description because this crosslinking condition is the middle value of the preferred range of this application, clearly demonstrating the influence of temperature and rotation speed on the microsphere performance throughout the preferred range, facilitating understanding by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Other temperatures and rotation speeds falling within the preferred range of this application are also within the scope of protection of this application, and will not be listed individually here.

[0062] In possible public examples, the centrifugation speed described in this application is preferably 2500~3500 rpm, and the centrifugation time is preferably 3~5 minutes, so as to balance the integrity of microspheres, collection efficiency, and purification effect. In this embodiment, a centrifugation speed of 3000 rpm and a time of 4 minutes are chosen for illustrative description because these centrifugation parameters are the middle values ​​of the preferred range of this application, clearly demonstrating the influence of centrifugation parameters on microsphere performance throughout the preferred range, facilitating understanding by those skilled in the art. However, this does not constitute any limitation on the scope of protection of this application. Other centrifugation parameters falling within the preferred range of this application are also within the scope of protection of this application, and will not be listed individually here.

[0063] In possible public examples, the freeze-drying time described in this application is preferably 24 hours, so that the microspheres can maintain sufficient network pore size and morphological integrity, taking into account both drying efficiency and microsphere structural integrity; at the same time, the temperature of the shaker is set to 45°C and the rotation speed is set to 800 rpm, so as to promote the rapid, uniform and efficient penetration of hydrophilic antitumor drugs into the internal network of microspheres without damaging the structural integrity and elastic properties of microspheres, thereby improving drug loading uniformity and drug loading capacity.

[0064] In possible public examples, the hydrophilic antitumor drug described in this application is preferably any one of pentafluorouracil, doxorubicin hydrochloride, and tetracycline hydrochloride, and the solvent of the impregnation solution is preferably sodium acetate buffer. Specifically, in this application embodiment, pentafluorouracil (5-FU) is selected as the drug and sodium acetate buffer as the impregnation solution solvent, thereby achieving good compatibility between the drug, solvent, and microsphere system. Pentafluorouracil, as a typical hydrophilic antitumor drug, has good water solubility compatible with sodium acetate buffer and excellent compatibility with the internal network of the elastic embolic microspheres, allowing for rapid penetration and stable loading. Sodium acetate buffer, as the solvent, maintains a suitable pH value in the system, preventing drug degradation and inactivation, ensuring drug loading stability, and promoting moderate swelling of the microspheres to improve drug loading and uniformity. This facilitates the understanding of the core technical logic of this application by those skilled in the art, but it does not constitute any limitation on the scope of protection of this application. Other drugs and solvents that can achieve the same or similar technical effects are also within the scope of protection of this application, and will not be listed individually here.

[0065] In possible public examples, the concentration of the hydrophilic antitumor drug in the impregnation solution described in this application is preferably 2-8 mg / mL, and the mass-to-volume ratio of the elastic embolic microspheres to the impregnation solution is preferably 4 mg:3 mL. The embodiments of this application use concentrations of 2 mg / mL, 4 mg / mL, and 6 mg / mL as representative examples because this group of concentrations uniformly covers the preferred range of 2-8 mg / mL, clearly demonstrating the influence of concentration on drug loading efficiency and drug distribution uniformity. Furthermore, each concentration is compatible with a solid-liquid ratio of 4 mg:3 mL, ensuring stable and efficient microsphere loading without drug waste or insufficient loading, while also considering representativeness and process repeatability. However, this does not constitute any limitation on the scope of protection of this application. Other concentrations falling within the preferred range of this application are also within the scope of protection of this application, and will not be listed individually here.

[0066] In possible disclosed examples, the microfluidic device of the microfluidic technology includes a quasi-two-dimensional polydimethylsiloxane microfluidic chip, and the channel structure of the polydimethylsiloxane microfluidic chip is a flow focusing structure. This application also provides a method for fabricating a microfluidic chip, preferably comprising:

[0067] Place a 3-inch silicon wafer at the center of the spin coater tray. Turn on the vacuum pump to allow it to adhere to the suction cup. Pour an appropriate amount of SU-8 2100 photoresist onto the center of the silicon wafer. Set the spin coater speed to 3000 rpm and the spin coater time to 40 seconds. Pre-bake at 65°C for 5 minutes, then at 95°C for 20 minutes. After natural cooling, place the mask on top, ensuring it is in complete contact with the photoresist. Expose under a mercury lamp for 24 seconds. Post-bake at 65°C for 5 minutes, then at 95°C for 15 seconds. After natural cooling, the chip is immersed in the developer (propylene glycol methyl ether acetate) and slowly shaken for 10 minutes; the chip positive mold is rinsed clean with isopropanol; the chip is placed on a heating stage at 150°C for 30 minutes to harden; the chip is then subjected to plasma treatment for 5 minutes, followed by chemical vapor deposition of 1H,1H,2H,2H-perfluorooctyltrichlorosilane for 24 hours to silanize it; the PDMS is poured onto a SU-8 mold and cured at 80°C for 4 hours; the PDMS is then cut and peeled off from the chip positive mold; holes are drilled to create a continuous phase inlet, a dispersed phase inlet, and a fluid outlet, thus obtaining the PDMS cover sheet; the PDMS substrate and the PDMS gasket without channel structure are bonded together after plasma treatment for 45 seconds to obtain a quasi-two-dimensional PDMS microfluidic chip with the following structure: Figures 1 to 2 As shown.

[0068] according to Figures 1 to 2 As can be seen, the microfluidic chip used in the embodiments of the present invention has a flow focusing structure and a channel height of 100μm.

[0069] Meanwhile, the geometric dimensions of the flow focusing point of the microfluidic chip channel used in the embodiments of the present invention are shown in Table 1.

[0070] Table 1: Geometric dimensions of the flow focal point in the microfluidic chip channel

[0071]

[0072] The second aspect of this application also provides drug-release elastic embolization microspheres prepared by the preparation method described in this application. The particle size of these drug-release elastic embolization microspheres is adjustable within the range of 15~80μm, and the particle size variation coefficient is less than 5%. This allows for precise adaptation to the embolization needs of different vascular sites, achieving targeted embolization without blocking normal peripheral blood vessels. It also avoids problems such as uneven embolization and blood flow disturbance caused by differences in particle size. At the same time, it ensures the consistency of drug loading and sustained-release performance of each microsphere. Combined with its elastic properties, it can further improve embolization stability and clinical application safety.

[0073] The third aspect of this application also provides the application of the drug-release elastic embolization microspheres described herein, specifically, the drug-release elastic embolization microspheres are used to prepare embolization preparations. The embolization preparations can be used for vascular interventional embolization therapy, and applicable conditions include, but are not limited to, malignant tumors, benign tumors, vascular malformations, and hemorrhagic diseases. Specifically, they can be used for interventional treatment of conditions such as liver cancer, lung cancer, uterine fibroids, hemangiomas, and ruptured esophageal varices.

[0074] The technical solution of this application will be further described below with reference to specific embodiments.

[0075] Example 1

[0076] This example provides a method for preparing drug-release elastic embolization microspheres, specifically including:

[0077] S1: Mix 0.1g of acetic acid with 4.9mL of deionized water to prepare a 2% wt acetic acid aqueous solution. Weigh 100mg of chitosan powder (degree of deacetylation 95%, viscosity 80mpa·s) and dissolve it in 5mL of acetic acid aqueous solution. Disperse the solution ultrasonically and stir overnight until the solution is clear to obtain a 2% w / v dispersed phase solution. At the same time, dissolve Span 80 in isopropyl palmitate to form a continuous phase with a Span 80 concentration of 4% w / v.

[0078] S2: After filtering the continuous phase and dispersed phase multiple times, transfer them separately into the syringe and push them out at a constant speed using a dual-channel injection pump. Maintain the continuous phase at a flow rate of 0.4 mL / min and the dispersed phase at a flow rate of 80 μL / h into the microfluidic chip. At the intersection of the two phases in the chip's microchannel, focus and shear to form a W / O monodisperse emulsion template.

[0079] S3: The genipin crosslinking agent was dissolved in a separately prepared continuous phase as the acceptor phase (genipin concentration was 2 mg / mL). The W / O monodisperse emulsion template prepared in the previous step was introduced into the acceptor phase. The crosslinking reaction was carried out at 40℃ and 50 rpm under magnetic stirring for 4, 6 and 8 hours, respectively. After the reaction was completed, the microspheres were centrifuged at 3000 rpm for 4 minutes and washed 3 times with ethanol and deionized water, respectively. Finally, the microspheres were freeze-dried for 24 hours to prepare the elastic embolic microspheres.

[0080] S4: Dissolve 5-fluorouracil (5-Fu) in sodium acetate buffer (0.01 mol / L, pH 5.5) to prepare an impregnation solution (impregnation solution concentration of 6 mg / mL). Add 8 mg of elastic embolic microspheres to 6 mL of the impregnation solution and shake at 800 rpm for 30 min in a constant temperature shaker at 45 °C to obtain drug sustained-release elastic embolic microspheres.

[0081] Figure 3 and Figure 4Scanning electron microscope (SEM) images of the surface and internal cross-section of the elastic embolization microspheres prepared in this example are shown, where images a-c represent crosslinking times of 4h, 6h, and 8h, respectively.

[0082] according to Figure 3 It can be seen that the elastic embolic microspheres prepared in this example are all regular spherical in shape, with uniform particle size and no adhesion, and an average particle size of 50 μm. Furthermore, the average particle size of the microspheres does not change significantly with prolonged cross-linking time, indicating that the particle size of the dried chitosan microspheres is determined solely by the close packing of chitosan molecular chains in the monodisperse W / O emulsion template. Increasing the degree of cross-linking only changes the tightness of the internal cross-linking of the microspheres, and has little effect on the particle size. According to... Figure 4 It can be seen that the internal structure of the elastic embolization microspheres prepared in this example is uniform, tightly cross-linked, and without obvious pores. The cross-linking network is looser closer to the inside of the microsphere, and the cross-linking structure inside the microsphere becomes tighter with the extension of cross-linking time. This indicates that by setting an appropriate cross-linking time, the network structure inside the elastic embolization microsphere can be effectively controlled, thereby promoting the improvement of drug loading capacity and long-term stable sustained-release performance.

[0083] Example 2

[0084] This example provides a method for preparing drug-release elastic embolic microspheres, specifically including:

[0085] S1: Mix 0.1g of acetic acid with 4.9mL of deionized water to prepare a 2% wt acetic acid aqueous solution. Weigh 100mg of chitosan powder (degree of deacetylation 95%, viscosity 80mpa·s) and dissolve it in 5mL of acetic acid aqueous solution. Disperse the solution ultrasonically and stir overnight until the solution is clear to obtain a 2% w / v dispersed phase solution. At the same time, dissolve Span 80 in isopropyl palmitate to form a continuous phase with a Span 80 concentration of 4% w / v.

[0086] S2: After filtering the continuous phase and dispersed phase multiple times, transfer them separately into the syringe and push them out at a constant speed using a dual-channel injection pump. Maintain the continuous phase at a flow rate of 0.4 mL / min and the dispersed phase at a flow rate of 80 μL / h into the microfluidic chip. At the intersection of the two phases in the chip's microchannel, focus and shear to form a W / O monodisperse emulsion template.

[0087] S3: The genipin crosslinking agent was dissolved in a separately prepared continuous phase as the acceptor phase (genipin concentrations were 4 mg / mL and 6 mg / mL, respectively), and the W / O monodisperse emulsion template prepared in the previous step was introduced into the acceptor phase. The crosslinking reaction was carried out at 40℃ and 50 rpm under magnetic stirring for 4 hours. After the reaction was completed, the microspheres were centrifuged at 3000 rpm for 4 minutes and washed 3 times with ethanol and deionized water, respectively. Finally, the microspheres were freeze-dried for 24 hours to prepare the elastic embolic microspheres.

[0088] S4: Dissolve 5-fluorouracil (5-Fu) in sodium acetate buffer (0.01 mol / L, pH 5.5) to prepare an impregnation solution (impregnation solution concentration of 6 mg / mL). Add 8 mg of elastic embolic microspheres to 6 mL of the impregnation solution and shake at 800 rpm for 30 min in a constant temperature shaker at 45 °C to obtain drug sustained-release elastic embolic microspheres.

[0089] Example 3

[0090] This example provides a method for preparing drug-release elastic embolic microspheres, specifically including:

[0091] S1: Mix 0.1g of acetic acid with 4.9mL of deionized water to prepare a 2% wt acetic acid aqueous solution. Weigh 100mg of chitosan powder (degree of deacetylation 95%, viscosity 80mpa·s) and dissolve it in 5mL of acetic acid aqueous solution. Disperse the solution ultrasonically and stir overnight until the solution is clear to obtain a 2% w / v dispersed phase solution. At the same time, dissolve Span 80 in isopropyl palmitate to form a continuous phase with a Span 80 concentration of 4% w / v.

[0092] S2: After filtering the continuous phase and dispersed phase multiple times, transfer them separately into the syringe and push them out at a constant speed using a dual-channel injection pump. Maintain the continuous phase at a flow rate of 0.4 mL / min and the dispersed phase at a flow rate of 20 μL / h into the microfluidic chip. At the intersection of the two phases in the chip's microchannel, focus and shear to form a W / O monodisperse emulsion template.

[0093] S3: The genipin crosslinking agent was dissolved in a separately prepared continuous phase as the acceptor phase (genipin concentration was 2 mg / mL). The W / O monodisperse emulsion template prepared in the previous step was introduced into the acceptor phase. The crosslinking reaction was carried out at 40℃ and 50 rpm under magnetic stirring for 4 hours. After the reaction was completed, the microspheres were centrifuged at 3000 rpm for 4 minutes and washed 3 times with ethanol and deionized water respectively. Finally, the microspheres were freeze-dried for 24 hours to prepare elastic embolic microspheres.

[0094] S4: Dissolve 5-fluorouracil (5-Fu) in sodium acetate buffer (0.01 mol / L, pH 5.5) to prepare an impregnation solution (impregnation solution concentration of 6 mg / mL). Add 8 mg of elastic embolic microspheres to 6 mL of the impregnation solution and shake at 800 rpm for 30 min in a constant temperature shaker at 45 °C to obtain drug sustained-release elastic embolic microspheres with a particle size of 43 μm.

[0095] Example 4

[0096] This example provides a method for preparing drug-release elastic embolic microspheres, specifically including:

[0097] S1: Mix 0.1g of acetic acid with 4.9mL of deionized water to prepare a 2%wt acetic acid aqueous solution. Weigh 150mg of chitosan powder (degree of deacetylation 95%, viscosity 80mpa·s) and dissolve it in 5mL of acetic acid aqueous solution. Disperse the solution ultrasonically and stir overnight until the solution is clear to obtain a 3%w / v dispersed phase solution. At the same time, dissolve Span 80 in isopropyl palmitate to form a continuous phase with a Span 80 concentration of 4%w / v.

[0098] S2: After filtering the continuous phase and dispersed phase multiple times, transfer them separately into the syringe and push them out at a constant speed using a dual-channel injection pump. Maintain the continuous phase at a flow rate of 0.4 mL / min and the dispersed phase at a flow rate of 20 μL / h into the microfluidic chip. At the intersection of the two phases in the chip's microchannel, focus and shear to form a W / O monodisperse emulsion template.

[0099] S3: The genipin crosslinking agent was dissolved in a separately prepared continuous phase as the acceptor phase (genipin concentration was 2 mg / mL). The W / O monodisperse emulsion template prepared in the previous step was introduced into the acceptor phase. The crosslinking reaction was carried out at 40℃ and 50 rpm under magnetic stirring for 4 hours. After the reaction was completed, the microspheres were centrifuged at 3000 rpm for 4 minutes and washed 3 times with ethanol and deionized water respectively. Finally, the microspheres were freeze-dried for 24 hours to prepare elastic embolic microspheres.

[0100] S4: Dissolve 5-fluorouracil (5-Fu) in sodium acetate buffer (0.01 mol / L, pH 5.5) to prepare an impregnation solution (impregnation solution concentration of 6 mg / mL). Add 8 mg of elastic embolic microspheres to 6 mL of the impregnation solution and shake at 800 rpm for 30 min in a constant temperature shaker at 45 °C to obtain drug sustained-release elastic embolic microspheres with a particle size of 53 μm.

[0101] To characterize the various properties of the elastic embolization microspheres prepared in this application, the following test examples 1 to 3 are provided.

[0102] Test Example 1

[0103] Weigh the elastic embolic microspheres prepared in this application and place them in a centrifuge tube. Add PBS buffer (pH 7.4) until the microspheres are completely submerged and shake well. Place the centrifuge tube in a constant temperature shaker at 37°C and shake at 600 rpm to ensure complete contact between the microspheres and the PBS buffer. Remove the tube after 24 hours. Take pictures of the microspheres before and after swelling using an inverted microscope, record and calculate the volume change of the microspheres before and after swelling, and calculate the swelling rate of the microspheres. The formula for calculating the swelling rate of the microspheres is as follows:

[0104]

[0105] In the formula, Swelling Rate is the swelling ratio, Ve is the average volume of the microspheres after swelling, and Vo is the average volume of the microspheres before swelling.

[0106] Figure 5 The swelling properties of the elastic embolic microspheres prepared in this application are shown as a function of genipinil concentration and crosslinking time.

[0107] according to Figure 5 It can be seen that the swelling ratio of the elastic embolization microspheres prepared in this application increases with the decrease of genipin concentration or the shortening of crosslinking time, indicating that changing the crosslinking agent concentration or crosslinking time will cause changes in the degree of crosslinking of the microspheres.

[0108] Test Example 2

[0109] This application utilizes a single-particle mechanical property testing system (SPFT2000, Yuaneng Technology (Xiamen) Co., Ltd.) to characterize elasticity. Specifically, a small amount of the elastic embolic microspheres prepared in this application is added to anhydrous ethanol and ultrasonically dispersed. The microsphere suspension is then dropwise added onto a glass slide provided with the instrument, and the slide is placed in a vacuum oven at 50°C for 24 hours to dry. The slide is then placed in the instrument, and a probe is used to vertically press down on a single microsphere at a speed of 1 μm / s, thereby obtaining the stress-strain curve of a single microsphere. The results are as follows: Figure 6 As shown; simultaneously, the Young's modulus of the microspheres was calculated by analyzing the curve, and the result is as follows. Figure 7 As shown.

[0110] according to Figure 7 It can be seen that, with the same crosslinking time, increasing the concentration of the crosslinking agent in the acceptor phase increases the Young's modulus of the microspheres. Before the emulsion template solidifies into a regular sphere (e.g., with a fixed genipin concentration of 2 mg / mL and a crosslinking time of less than 4 h), extending the crosslinking time increases the Young's modulus of the microspheres. Combined with the swelling performance test of the microspheres, it can be seen that when the concentration of the crosslinking agent in the acceptor phase is fixed, extending the crosslinking time increases the crosslinking density of the microspheres, but the Young's modulus of the microspheres does not change significantly.

[0111] Test Example 3

[0112] 3.1 Drug loading rate

[0113] The absorbance of the impregnation solution before and after drug loading was measured using a UV-Vis spectrophotometer. Based on the UV standard curve of 5-Fu / sodium acetate, the drug content adsorbed into the elastic embolic microspheres prepared in Example 1 was calculated, and the results were recorded in Table 2.

[0114] Table 2: Drug loading rate at different cross-linking times

[0115]

[0116] As shown in Table 2, when the crosslinking time increased from 4h to 8h, the drug loading rate (DL) of the microspheres decreased from 7.04±0.75% to 19.39±1.53%.

[0117] 3.2 Drug release kinetics

[0118] PBS buffer solutions with pH values ​​of 6.8 and 7.4 were used as release media. 8 mg of the elastic embolic microspheres prepared in Example 1 were weighed and dispersed in centrifuge tubes containing 2 mL of release media. The tubes were placed in a 37°C constant-temperature shaker at 600 rpm. At the set time points, 400 μL of release media was collected, and 400 μL of blank PBS buffer was added simultaneously. The absorbance was measured at 266 nm using a UV spectrophotometer. The drug content in the release media was determined using a standard curve. Finally, a drug release curve was calculated and plotted. The results are as follows: Figure 8 As shown.

[0119] according to Figure 8 It is known that the elastic embolic microspheres exhibit a rapid drug release rate in the first 100 minutes, which then plateaus. The release rate of the elastic embolic microspheres is faster in a medium with a pH of 6.8 than in a medium with a pH of 7.4, and the cumulative drug release is also higher, demonstrating a significant pH-responsiveness. Furthermore, the longer the cross-linking time of the microspheres, the higher the drug release rate and the higher the cumulative release amount of the sustained-release elastic embolic microspheres. Therefore, considering both the elasticity of the embolic microspheres and the sustained-release properties of the drug, the preferred cross-linking time in this application is 4–8 hours.

[0120] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0121] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A method for preparing drug-release elastic embolic microspheres, characterized in that, Includes the following steps: Chitosan powder was dissolved in an aqueous acetic acid solution and treated to obtain a clear dispersed phase solution; The dispersed phase solution and the continuous phase are mixed using microfluidics to form a W / O monodisperse emulsion template, wherein the continuous phase comprises organic oil and W / O type surfactant; Genipin crosslinking agent was dissolved in organic oil containing W / O type surfactant to form an acceptor phase. The W / O monoemulsion template was introduced into the acceptor phase and crosslinked for 4 to 24 hours under magnetic stirring. After the crosslinking reaction was completed, the obtained microspheres were centrifuged, washed and freeze-dried in sequence to obtain elastic embolization microspheres. The elastic embolic microspheres are dispersed in an impregnation solution containing a hydrophilic antitumor drug and then shaken in a shaker to obtain drug-release elastic embolic microspheres.

2. The preparation method according to claim 1, characterized in that, The crosslinking time under magnetic stirring is 4 to 8 hours.

3. The preparation method according to claim 1, characterized in that, The chitosan has the following characteristics: Deacetylation degree >95%; Viscosity is 50~100 mPa·s; Furthermore, the mass-volume ratio of the chitosan to the acetic acid aqueous solution is 1~3% w / v.

4. The preparation method according to claim 3, characterized in that, The W / O type surfactant is selected from one or more of Span 85 and Span 80; And / or, the organic oil is selected from one or more of isopropyl palmitate, soybean oil, liquid paraffin, dimethyl silicone oil and petroleum ether.

5. The preparation method according to claim 1, characterized in that, The continuous phase and the receiving phase each contain a W / O type surfactant with a mass-to-volume ratio of 4-6% w / v to organic oil, and the receiving phase contains a genipin crosslinking agent with a mass-to-volume ratio of 2-6 mg / mL.

6. The preparation method according to claim 1, characterized in that, The temperature of the magnetic stirring is maintained at 35~45℃, and the rotation speed is 50rpm. And / or, the centrifugation speed is set to 2500~3500 rpm, and the centrifugation time is 3~5 minutes; And / or, the freeze-drying time is 24 hours; And / or, the temperature of the shaker is set to 45°C and the rotation speed is set to 800 rpm.

7. The preparation method according to claim 1, characterized in that, The hydrophilic antitumor drug is selected from any one of pentafluorouracil, doxorubicin hydrochloride, and tetracycline hydrochloride; And / or, the solvent of the impregnation solution contains sodium acetate buffer; The concentration of the hydrophilic antitumor drug in the impregnation solution is 2-8 mg / mL, and the mass-to-volume ratio of the elastic embolic microspheres to the impregnation solution is 4 mg:3 mL.

8. The preparation method according to claim 1, characterized in that, The microfluidic device of the microfluidic technology includes a quasi-two-dimensional polydimethylsiloxane microfluidic chip, and the channel structure of the polydimethylsiloxane microfluidic chip is a flow focusing structure.

9. A drug-release elastic embolic microsphere prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The particle size of the drug-release elastic embolization microspheres is adjustable within the range of 15~80μm, and the particle size variation coefficient is less than 5%.

10. The use of a drug sustained-release elastic embolization microsphere prepared by any one of claims 1 to 8 in the preparation of embolization formulations.

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