A method for electrospinning preparation of labelable radionuclide chitosan hydrogel microspheres

Abstract

The present application relates to the technical field of biological medicine materials, and relates to an electrospinning manufacturing technology of chitosan hydrogel microspheres capable of being labeled with radionuclides. The core of the method is that: first, through electrostatic spinning (electrospinning) technology, chitosan and an anion crosslinking agent (such as tripolyphosphate) are reacted under specific parameters (voltage 1-20 kV, flow rate 10-100 μl / min) to prepare chitosan hydrogel microspheres with uniform particle size and solid structure, and the particle size of the chitosan hydrogel microspheres can be accurately controlled in the optimal application range of 30 μm to 300 μm through process parameters. The obtained hydrogel microspheres have the characteristics of interventional embolic agents and reaction grafting with chelating agents (such as SHPP and DOTA) containing specific functional groups.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a chitosan chelated hydrogel microsphere that can be labeled with radionuclides and its preparation method. Background Technology

[0002] In the global public health field, the incidence and mortality rates of cancer continue to rise, making it one of the leading malignant diseases threatening human life and health. Despite continuous advancements in clinical treatment methods, traditional treatments (such as surgical resection, chemotherapy, and conventional radiotherapy) still have limitations: surgery is highly invasive and carries a high risk of postoperative recurrence; chemotherapy and conventional radiotherapy lack targeting and easily cause severe damage to normal tissues, leading to a series of side effects such as decreased immunity and organ dysfunction, significantly reducing treatment tolerance and quality of life. Therefore, developing novel cancer treatment strategies that are highly efficient, minimally invasive, highly targeted, and minimize damage to normal tissues has become an urgent need in the biomedical field.

[0003] Radionuclide therapy, as a highly promising minimally invasive treatment technique, has gained widespread attention and application in clinical cancer treatment due to its advantages such as ease of operation, controllable treatment costs, and significant local tumor-killing effects. Its core treatment principle utilizes ionizing radiation such as gamma rays and beta rays released by radionuclides to precisely target the DNA of tumor cells, inhibiting tumor cell proliferation or directly inducing apoptosis, thereby achieving the therapeutic goal of inhibiting or eliminating tumor growth. However, traditional systemic radionuclide therapy faces a core technical bottleneck: radionuclides lack specific distribution capabilities in the body. While targeting tumor lesions, they indiscriminately diffuse into surrounding normal tissues and organs, causing serious side effects such as bone marrow suppression, gastrointestinal reactions, and liver and kidney damage. This problem not only reduces patient compliance but may even lead to treatment interruption due to exacerbated side effects, severely impacting overall treatment efficacy and limiting the clinical application boundaries of radionuclide therapy.

[0004] The key to overcoming the aforementioned technical bottlenecks lies in constructing an ideal radionuclide carrier system. This carrier must achieve the core functions of "targeted delivery, in-situ retention, and stable release," while simultaneously meeting multiple performance requirements: First, it must possess excellent biocompatibility to avoid triggering immune rejection or toxicity accumulation in vivo, ensuring treatment safety. Second, it must have good biodegradability, allowing it to be naturally metabolized by the body after completing its therapeutic mission, reducing the potential risks associated with long-term retention. Third, it must be able to efficiently and stably load and label radionuclides, preventing their shedding and diffusion within the body and ensuring that radiation energy is concentrated on the tumor lesion. Fourth, it must possess controllable physicochemical properties, such as particle size uniformity and structural stability, to adapt to different administration methods (such as interventional embolization and local injection) and the treatment needs of different types of tumor lesions.

[0005] Among numerous carrier materials, hydrogels have become a hot research area for radionuclide carriers due to their three-dimensional network cross-linked structure, high water content (close to human tissue fluid), and good biocompatibility. Compared with ordinary bulk hydrogels, microsphere hydrogels exhibit more prominent performance advantages: ① The abundant active groups on the surface of microspheres can form non-covalent interactions with the matrix material, significantly enhancing the structural stability of the carrier system and preventing degradation or breakage during in vivo transport; ② Microspheres can serve as physical cross-linking sites, further increasing the cross-linking density of the composite carrier, optimizing the mechanical strength and radionuclide retention capacity of the carrier, and reducing radionuclide leakage; ③ The microsphere structure has a high specific surface area and porous characteristics, which can significantly improve the loading capacity and labeling efficiency of radionuclides under the same water content conditions, while enabling slow and continuous release of radionuclides at the lesion site, prolonging the radiation therapy time and improving the treatment effect. These unique advantages make microsphere hydrogels irreplaceable in the fields of targeted drug delivery and interventional tumor therapy.

[0006] Chitosan, as a natural polysaccharide biomaterial, is widely available, exhibits excellent biocompatibility, and produces non-toxic degradation products. Furthermore, its molecular chain is rich in active groups such as amino and hydroxyl groups, making it easy to chemically modify and cross-link, thus making it an ideal substrate for constructing hydrogel carriers. However, existing technologies for preparing chitosan-based radionuclide carriers still have several shortcomings: some preparation processes are complex and time-consuming, making it difficult to meet the large-scale production requirements for clinical translation; the prepared microspheres have poor particle size uniformity, failing to accurately adapt to the particle size requirements of clinical treatment scenarios such as tumor vascular embolization; the radionuclide labeling efficiency is low and the stability is insufficient, easily leading to radionuclide shedding and diffusion problems; some carriers lack good biodegradability, and long-term retention in the body may cause inflammatory reactions or tissue damage. In addition, existing carriers are mostly designed for single radionuclides, lacking versatility and failing to meet diverse clinical treatment needs.

[0007] Therefore, developing a chitosan-based microsphere carrier that is simple, efficient, safe, and scalable, capable of producing controllable particle size, high radionuclide labeling efficiency, strong stability, and excellent biocompatibility, is of significant practical and clinical value for overcoming the technical bottlenecks of traditional radionuclide therapy, improving the targeting and safety of treatment, and promoting the clinical translation and development of radionuclide therapy technology. Furthermore, the expanded application of this type of carrier in scenarios such as interventional embolization therapy for tumors provides a new technical pathway for multimodal tumor treatment. Summary of the Invention

[0008] The purpose of this invention is to provide an electrospinning method for preparing chitosan chelated hydrogel microspheres that can be labeled with radionuclides. This method is simple, efficient, safe, and can be mass-produced. It can also produce chitosan-based microsphere carriers with controllable particle size, high radionuclide labeling efficiency, strong stability, and excellent biocompatibility.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing chitosan chelated hydrogel microspheres that can be labeled with radionuclides. The method uses electrospinning to prepare chitosan-based hydrogel microspheres, including electrospinning chitosan and crosslinking substances to prepare chitosan hydrogel microspheres. The cross-linking substance is selected from at least one of the following groups: tripolyphosphate, alginate, pectin, carboxymethyl cellulose, polyglutamic acid, protein, DNA, and RNA. The parameters of the electrospinning process are: chitosan concentration 1.0-5.0%, needle diameter 30-100μm, flow rate 10-100μL / min, applied voltage 5-15kV, and working distance 8-15cm. By adjusting the above process parameters, the particle size and physical properties of microspheres can be controlled. The formation of microspheres is determined by the combination of the above variables.

[0010] Furthermore, the particle size of the microspheres exhibits the following trend as process parameters change: (1) As the flow rate increases, the particle size tends to increase; (2) As the applied voltage increases, the particle size tends to decrease or the particle uniformity changes; (3) As the concentration of chitosan increases, the particle size increases and the structural stability is enhanced.

[0011] Furthermore, the electrospinning process is affected by environmental conditions such as temperature and humidity, and stable process operation requires maintaining consistent environmental conditions. Under the same process conditions, slight differences in the environment may cause changes in the particle size distribution of microspheres; Microspheres within a specific size range can be screened out through a post-screening process.

[0012] Furthermore, the corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 30–60 μm are as follows: chitosan concentration 1.0–5.0%, needle diameter 30–60 μm, flow rate 10–30 μL / min, applied voltage 8–15 kV, and working distance 8–15 cm.

[0013] Furthermore, the corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 60–150 μm are as follows: chitosan concentration 1.0–5.0%, needle diameter 30–100 μm, flow rate 20–60 μL / min, applied voltage 8–15 kV, and working distance 8–15 cm.

[0014] Furthermore, the corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 150–300 μm are as follows: chitosan concentration 1.0–5.0%, needle diameter 80–150 μm, flow rate 30–100 μL / min, applied voltage 5–12 kV, and working distance 8–15 cm.

[0015] Furthermore, the process also includes a microsphere purification step: the prepared microspheres are purified using a polymer network with pore sizes smaller than the microsphere particle size to remove unreacted raw materials and crush particle impurities; the polymer network includes a 50μm-150μm nylon sieve.

[0016] The present invention also provides a chitosan chelated hydrogel microsphere that can be labeled with radionuclides, prepared by the aforementioned preparation method.

[0017] Furthermore, the microspheres have a uniform and controllable particle size and can be chelated with various chelating agents for labeling different radionuclides; The chelating agent is at least one selected from the group consisting of N-succinimide-3-(4-hydroxyphenyl)propionate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, diethylenetriaminepentaacetic acid, histidine, tyrosine, and proteins containing tyrosine; wherein, when the chelating agent is N-succinimide-3-(4-hydroxyphenyl)propionate, iodo-chitosan chelated hydrogel microspheres are obtained; when the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, lutetium-based chitosan chelated hydrogel microspheres are obtained.

[0018] Furthermore, the microspheres can be used as arterial embolization agents for interventional radiotherapy, and after being labeled with radionuclides, they can be used for internal radiation embolization therapy.

[0019] The beneficial effects of this invention are as follows: The chitosan hydrogel microspheres labeled with radionuclides and their preparation method provided by this invention have significant advantages in terms of technology and process, effectively solving the core pain points in the preparation of existing radionuclide carriers. First, the preparation process is scientifically and efficiently designed. Second, the particle size can be precisely controlled. By adjusting the chitosan concentration (1%-5% mass fraction), electrospinning voltage (1kV-20kV), and flow rate (10-100 μl / min), the microsphere particle size can be precisely controlled to the optimal application range of 30μm-300μm. Furthermore, increasing the chitosan concentration can further improve the density, robustness, and size uniformity of the hydrogel particles, providing flexible support for adapting to different clinical treatment scenarios (such as embolization therapy). Third, the product has excellent purity and stability. The selection of cross-linking substances (such as tripolyphosphate, alginate, etc.) can not only enhance the stability of the hydrogel structure, but also improve the stability of radionuclide labeling and prevent the radionuclide from spreading to normal tissue outside the lesion. Subsequent purification through a polymer network with a mesh size smaller than that of microspheres (such as a 50μm nylon screen) can effectively remove impurities such as unreacted raw materials and crushed particles, ensuring the high purity of the final product.

[0020] The technical solution of this invention demonstrates outstanding value in clinical applications, providing crucial support for the optimization and upgrading of embolization therapy technology. On one hand, the chitosan hydrogel microspheres possess excellent biocompatibility and potential biodegradability, allowing for safe metabolism in vivo and avoiding the risks of immune rejection or toxicity accumulation caused by long-term retention, thus significantly improving treatment safety. On the other hand, its platform-based design is adaptable to various radionuclides, and the high specific surface area and porous structure of the microspheres enhance the nuclide loading capacity. Furthermore, these microspheres are suitable for embolization treatment scenarios and possess the characteristic of labeling radionuclides, expanding the scope of clinical applications. They provide core materials for developing highly efficient and minimally invasive novel cancer and embolization treatment agents, which is of great significance for promoting technological progress and improving clinical treatment efficacy in the field of biomedical materials. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a microscopic image of the 30-40 μm chitosan hydrogel in Example 1 of the present invention; Figure 2 This is a microscopic image of the 60-150 μm chitosan hydrogel in Example 1 of the present invention; Figure 3This is a microscopic image of the 150-300 μm chitosan hydrogel in Example 1 of the present invention; Figure 4 These are optical microscope images of the 1% and 3% chitosan hydrogels in Experimental Example 1 of this invention; Figure 5 Microscopic images of chitosan hydrogels prepared under different electrospinning parameters in Experimental Example 2 of this invention; Figure 6 This is a diagram showing the adverse phenomena under adverse conditions in the experimental examples of this invention. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The specific ranges of the key parameters involved in the preparation process of this invention are as follows: Chitosan concentration: can be adjusted according to needs, with a typical range of 1%-3% (mass fraction). The higher the concentration, the better the hydrogel particle density, robustness and stability, and the more uniform the particle size. Electrospinning parameters: voltage range is 1kV-20kV, with a preferred range of 5kV-12kV; flow rate range is 10-100 μl / min. By adjusting the voltage and flow rate, the hydrogel particle size can be controlled to the optimal application range of 30μm-300μm. The present invention will be further illustrated below through examples.

[0029] Example 1: Preparation and Model of Chitosan Hydrogel 1.1 In this embodiment, chitosan hydrogels were prepared by fixing the chitosan concentration and combining it with specific electrospinning parameters. The specific steps are as follows: (1) Preparation of 1% chitosan hydrogel Weigh 25 mg of chitosan and dissolve it in 2.5 mL of 0.1 M acetic acid solution to prepare a 1% chitosan solution. Separately, take 5 g of sodium tripolyphosphate (TPP) and dissolve it in a solution using a pump to form a crosslinking substance. Electrospin the chitosan solution and sodium tripolyphosphate solution under conditions of 10 kV voltage and 0.02 mL / min to prepare a 1% chitosan hydrogel.

[0030] (2) Preparation of 2% chitosan hydrogel Weigh 50 mg of chitosan and dissolve it in 2.5 mL of 0.1 M acetic acid solution to prepare a 2% chitosan solution. Separately, take 5 g of sodium tripolyphosphate (TPP) and dissolve it in a solution using a pump to form a crosslinking substance. Electrospin the chitosan solution and sodium tripolyphosphate solution under conditions of 12 kV voltage and 0.02 mL / min to prepare a 2% chitosan hydrogel.

[0031] 1.2 Chitosan hydrogel particle size models (1) 30-40μm chitosan hydrogel model Chitosan concentration: 1.0~5.0%, needle diameter: 30~60μm, flow rate: 10~30μL / min, applied voltage: 8~15KV, working distance: 8~15cm. Figure 1 ) (2) 60-150μm chitosan hydrogel model Chitosan concentration: 1.0~5.0%, needle diameter: 30~100μm, flow rate: 20~60μL / min, applied voltage: 8~15KV, working distance: 8~15cm. Figure 2) (3) 150-300μm chitosan hydrogel model Chitosan concentration: 1.0~5.0%, needle diameter: 80~150μm, flow rate: 30~100μL / min, applied voltage: 5~12KV, working distance: 8~15cm. Figure 3 ) Experiment Example 1: Comparison of Particle Size and Morphology Based on Chitosan Concentration 1. Experimental Objective The particle size and morphological characteristics of chitosan hydrogels prepared at different chitosan concentrations were compared.

[0032] 2. Experimental subjects Example 1 (1) prepared 1% chitosan hydrogel, Example 1 (2) prepared 2% chitosan hydrogel.

[0033] 3. Experimental Methods The particle morphology of the two chitosan hydrogels was observed at 40x magnification using an optical microscope, and the uniformity of particle size was evaluated.

[0034] 4. Experimental Results Observation results as follows Figure 4 As shown, by Figure 4 It can be seen that, compared with 1% chitosan hydrogel (left), 2% chitosan hydrogel (right) has smooth spherical particles, higher particle density, and more uniform particle size.

[0035] Experiment 2: Adjusting the particle size of chitosan hydrogel by changing electrospinning conditions 1. Experimental Objective To investigate the regulatory effects of electrospinning voltage and flow rate on the particle size of 2% chitosan hydrogel.

[0036] 2. Experimental subjects Chitosan hydrogels were prepared by adjusting the electrospinning parameters using the 2% chitosan solution from Example 1 as raw material.

[0037] 3. Experimental Methods Keeping the chitosan concentration at 2% and the crosslinking agent at sodium tripolyphosphate, and other conditions constant, two sets of electrospinning parameters were set: Voltage 12kV, flow rate 10μl / min; Voltage 11kV, flow rate 10μl / min; After preparing two types of chitosan hydrogels, the average particle size was observed and measured.

[0038] 4. Experimental Results In the preparation of chitosan hydrogels, the particle size changes with the combination of parameters, and the following general pattern is observed: when the flow rate increases, the particle size tends to increase; when the applied voltage increases, the particle size tends to decrease, or the particle uniformity changes; when the chitosan concentration increases, the particle size increases, and the structural stability of the particles is enhanced.

[0039] Experimental results are as follows Figure 5 As shown, by Figure 5 It can be seen that under the conditions of 12kV voltage and 0.01mL / min flow rate, the average particle size of the generated chitosan hydrogel is 40μm (left); under the conditions of 11kV voltage and 0.01mL / min flow rate, the average particle size of the generated chitosan hydrogel is 70μm (right), indicating that the particle size of chitosan hydrogel can be precisely controlled by adjusting the electrospinning voltage and flow rate.

[0040] Adverse phenomena occurring under adverse conditions ( Figure 6 ) Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing chitosan chelated hydrogel microspheres capable of labeling radionuclides, characterized in that, Chitosan-based hydrogel microspheres were prepared by electrospinning, including electrospinning chitosan and crosslinking substances to prepare chitosan hydrogel microspheres. The cross-linking substance is selected from at least one of the following groups: tripolyphosphate, alginate, pectin, carboxymethyl cellulose, polyglutamic acid, protein, DNA, and RNA. The parameters of the electrospinning process are: chitosan concentration 1.0-5.0%, needle diameter 30-100μm, flow rate 10-100μL / min, applied voltage 5-15kV, and working distance 8-15cm. By adjusting the above process parameters, the particle size and physical properties of microspheres can be controlled. The formation of microspheres is determined by the combination of the above variables.

2. The preparation method according to claim 1, characterized in that, The particle size of the microspheres exhibits the following trend as process parameters change: (1) As the flow rate increases, the particle size tends to increase; (2) As the applied voltage increases, the particle size tends to decrease or the particle uniformity changes; (3) As the concentration of chitosan increases, the particle size increases and the structural stability is enhanced.

3. The preparation method according to claim 1, characterized in that, The electrospinning process is affected by temperature and humidity conditions, and stable operation requires maintaining consistent environmental conditions. Under the same process conditions, slight differences in the environment may cause changes in the particle size distribution of microspheres; Microspheres within a specific size range can be screened out through a post-screening process.

4. The preparation method according to claim 1, characterized in that, The corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 30-60 μm are: chitosan concentration 1.0-5.0%, needle diameter 30-60 μm, flow rate 10-30 μL / min, applied voltage 8-15 kV, and working distance 8-15 cm.

5. The preparation method according to claim 1, characterized in that, The corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 60–150 μm are: chitosan concentration 1.0–5.0%, needle diameter 30–100 μm, flow rate 20–60 μL / min, applied voltage 8–15 kV, and working distance 8–15 cm.

6. The preparation method according to claim 1, characterized in that, The corresponding process parameters for preparing chitosan hydrogel microspheres with a particle size of 150–300 μm are: chitosan concentration 1.0–5.0%, needle diameter 80–150 μm, flow rate 30–100 μL / min, applied voltage 5–12 kV, and working distance 8–15 cm.

7. The preparation method according to any one of claims 1 to 6, characterized in that, It also includes a microsphere purification step: the prepared microspheres are purified using a polymer network with pores smaller than the microsphere particle size to remove unreacted raw materials and crushed particle impurities; the polymer network includes a 50μm-150μm nylon sieve.

8. A chitosan chelated hydrogel microsphere capable of labeling radionuclides, characterized in that, It is prepared by any one of claims 1 to 7.

9. The chitosan chelated hydrogel microspheres according to claim 8, characterized in that, The microspheres have a uniform and controllable particle size and can be chelated with a variety of chelating agents to label different radionuclides. The chelating agent is at least one selected from the group consisting of N-succinimide-3-(4-hydroxyphenyl)propionate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, diethylenetriaminepentaacetic acid, histidine, tyrosine, and proteins containing tyrosine; wherein, when the chelating agent is N-succinimide-3-(4-hydroxyphenyl)propionate, iodo-chitosan chelated hydrogel microspheres are obtained; when the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, lutetium-based chitosan chelated hydrogel microspheres are obtained.

10. The chitosan chelated hydrogel microspheres according to claim 8 or 9, characterized in that, The microspheres can be used as arterial embolization agents for interventional radiotherapy, and can be used for internal radiation embolization therapy after being labeled with radionuclides.

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