A one-step chitosan-grafted iodhexol autoradiographic microsphere, its preparation method and application
By crosslinking chitosan and iohexol with genipin in a one-step process to form stable self-developing microspheres, the problems of complex traditional two-step processes and easy developer detachment are solved, achieving synergistic optimization of development and structure and improved biocompatibility.
Patent Information
- Application Number
- CN202610229119.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-26
- Publication Date
- 2026-06-30
Smart Images
Figure CN122297753A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical materials technology, specifically to an autoradiographic embolization microsphere for medical diagnosis and treatment, and more particularly to an autoradiographic microsphere made by one-step grafting of chitosan with iohexol, its preparation method and application. Background Technology
[0002] Interventional embolization is a core treatment for vascular diseases such as tumors. It involves injecting embolic materials into diseased blood vessels to block nutrient supply and inhibit lesion growth, requiring imaging guidance and monitoring throughout the process. Self-illuminating microspheres, as a core material for integrated diagnosis and treatment, have significant application value in medical fields such as tumor embolization and tissue repair, and must simultaneously meet the requirements of clear imaging, good biocompatibility, and functional stability. Chitosan, as a natural polyaminopolysaccharide, possesses excellent biocompatibility and biodegradability, making it an ideal medical carrier material. However, chitosan alone lacks imaging function and requires grafting with contrast agents to achieve self-illuminating properties. Iohexol, a commonly used non-ionic iodine contrast agent in clinical practice, features high imaging sensitivity and low toxicity, and is frequently used in the preparation of self-illuminating materials.
[0003] In traditional techniques, the combination of chitosan and iohexol often employs a two-step "graft-crosslinking" method. First, chemical modification is used to graft the two materials, followed by the addition of a crosslinking agent for curing. This method suffers from problems such as complex processes, low reaction efficiency, and easy detachment of the developing agent. Furthermore, the crosslinking agents commonly used in existing technologies are often chemically synthesized reagents such as glutaraldehyde, which have poor biocompatibility and are prone to inducing inflammatory reactions, thus limiting the clinical application of the materials.
[0004] Genipin, as a natural plant extract, possesses advantages such as good biocompatibility and low cytotoxicity. Its cross-linking reaction with chitosan follows the Schiff reaction mechanism, resulting in a stable cross-linked structure. However, there are currently no reports on the one-step preparation of self-developing microspheres using genipin as a cross-linking agent to achieve integrated grafting and cross-linking of chitosan and iohexol. Therefore, developing a self-developing microsphere with a simple process, excellent biocompatibility, and balanced development and structural properties has significant practical application value. Summary of the Invention
[0005] To overcome the shortcomings of existing technologies, this invention provides a one-step method for preparing chitosan self-illuminating embolic microspheres. Chitosan is used as the carrier skeleton, iohexol is used as the self-illuminating functional component, and genipin, a natural extract, is used as the crosslinking agent. The grafting and crosslinking curing of chitosan and iohexol are achieved in one step to form self-illuminating embolic microspheres with uniform particle size, stable structure, and high mechanical strength.
[0006] The above-mentioned objective of this invention is achieved through the following technical solution: A self-developing microsphere grafted with iohexol using a one-step method is described. Chitosan serves as the carrier framework, iohexol is the self-developing functional component, and genipin, a natural extract, is used as the cross-linking agent. The grafting and cross-linking curing of chitosan and the developer are achieved in one step, forming self-developing embolic microspheres with uniform particle size, stable structure, and high mechanical strength.
[0007] A one-step method for preparing self-developing microspheres grafted with iodhexol onto chitosan, the specific steps of which are as follows: S1. Add chitosan to acetic acid solution and stir at room temperature for 10-30 min until it is completely dissolved; add iodhexol and stir at room temperature for 10-30 min until it is completely dissolved to obtain chitosan-iodhexol mixed solution. S2 Add the mixed solution obtained in S1 to liquid paraffin containing emulsifier and stir at high speed for 10-30 min to form a stable W / O type emulsion; S3 Add genipin solution dropwise to the emulsion, stir at a constant temperature, and achieve grafting and cross-linking curing in one step; After the S4 reaction was completed, the microspheres were centrifuged and washed with deionized water containing 0.5-1.0% (w / v) Tween 80 to remove the liquid paraffin on the microspheres. The microspheres were then eluted and dehydrated using a gradient of 30%-50%-70%-95% ethanol and dried under vacuum at room temperature to obtain blue chitosan autoradiographic embolization microspheres.
[0008] Furthermore, the microspheres have a particle size of 100~1000μm, a CT value ≥300 HU, a compressive strength ≥0.5 MPa, and a mass retention rate ≥90% after immersion in simulated body fluid for 30 days.
[0009] Furthermore, in step S1, iohexol is a commonly used water-soluble contrast agent in clinical practice, and can also be replaced by water-soluble iodine-containing contrast agents such as iopamidol or meglumine diatrizoate, with a concentration of 2.0~8.0 mg / mL; the concentration of the chitosan solution is 10~40 mg / mL.
[0010] Furthermore, in step S2, the dispersion medium is liquid paraffin, or one or more mixtures of edible oils such as soybean oil or olive oil; the emulsifier is one or more combinations of dehydrated sorbitan fatty acid ester surfactants such as Span-80, and the emulsifier concentration is 0.5~5 wt% (w / v); the volume ratio of the aqueous phase to the oil phase is 1:5~20; and the stirring speed is 1000~5000 rpm.
[0011] Furthermore, in step S3, the concentration of the genipin solution is 1.0~4.0 mg / mL, the volume ratio of the added volume to the chitosan solution is 1:10~20, the dropping rate is 0.5~2.0 mL / min, the stirring speed is maintained at 1000~3000 rpm, the cross-linking reaction time is 3~6 h, and the reaction temperature is 35~45℃.
[0012] Another objective of this invention is to protect the application of autoradiographic microspheres in medical imaging tracing.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Process Innovation: Breaking through the limitations of the traditional two-step "grafting-crosslinking" method, a one-step process was developed, which simultaneously completes the grafting reaction of chitosan and iohexol and the crosslinking and curing of genipin in the emulsion system, simplifying the process, improving the stability of developer loading, significantly increasing production efficiency, and reducing industrialization costs.
[0014] 2. Innovative material formulation: The precise formulation of natural biocompatible materials (chitosan, genipin) with commonly used clinical contrast agents (iohexol) achieves synergistic optimization of imaging function, biosafety and structural stability.
[0015] 3. Innovative performance control: By synergistically controlling the raw material ratio and process parameters, the microsphere particle size, iodine loading and imaging effect are precisely matched to meet the application needs of different medical scenarios.
[0016] 4. Excellent performance: The microspheres have stable iodine loading, clear development, good biocompatibility, and are biodegradable, solving the problem of imbalance between development and biological performance in traditional materials.
[0017] 5. Safe and environmentally friendly: It uses natural genipin instead of chemical cross-linking agents, with no release of toxic or harmful substances. The preparation process is mild and environmentally friendly.
[0018] 6. Wide range of applications: It can be used for real-time tracking in tumor embolization therapy, in vivo positioning of tissue engineering scaffolds, and other scenarios, realizing integrated diagnosis and treatment, with broad application prospects. Attached Figure Description
[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0020] Figure 1 Chitosan microspheres of different sizes: a = 242 μm, b = 353 μm, c = 478 μm, and d = 611 μm. Figure 2 The X-ray self-illumination properties of the microspheres are shown in Figure 1. A represents chitosan microspheres, and B represents chitosan self-illuminating microspheres. Detailed Implementation
[0021] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.
[0022] Example 1: Preparation of chitosan microspheres by emulsification method 1. Microsphere preparation process Chitosan was prepared into solutions of different concentrations by adding it to acetic acid solution. 10 mL of the chitosan solution was added to 60 mg of iohexol, and the mixture was stirred at room temperature for 30 min (200 rpm) until completely dissolved, yielding a chitosan-iohexol mixed solution. 10 mL of the above chitosan-iohexol mixed solution was slowly added to 100 mL of liquid paraffin containing 1.0% (w / v) span 80, and the mixture was stirred at 1000 rpm for 30 min to form a stable W / O emulsion. 4 mL of genipin solution with a concentration of 1 mg / mL was added dropwise to the emulsion at a rate of 0.5 mL / min, with continuous stirring (1000 rpm). The mixture was reacted at 40℃ in the dark for 4 h, allowing genipin to crosslink the amino groups on the chitosan and iohexol to obtain water-containing self-developing microspheres. After the reaction was complete, the mixture was centrifuged, and the microspheres were separated using a solution containing 1.0% (w / v) span 80. The microspheres were washed three times with Tween 80 deionized water to remove liquid paraffin from their surface; they were then eluted and dehydrated using a gradient of 30%-50%-70%-95% ethanol, and vacuum dried at room temperature to obtain blue chitosan autoradiographic embolization microspheres.
[0023] 2. Performance Testing (1) Measuring the particle size of microspheres At least 100 microsphere images were obtained by randomly selecting 10 fields of view under an inverted microscope. The particle size of each microsphere was read using ImageJ software, and the average value was calculated as D0.
[0024] (2) Measure compressive strength A certain amount of uniformly dispersed hydrogel microspheres were filled into a cylindrical mold, and axial compression was performed using a universal testing machine. The stress-strain curves were recorded. The stress value at which the microspheres underwent plastic deformation or a sudden stress change was used as the reference value for the average compressive strength of the batch of microspheres.
[0025] 3. Test Results The above experimental results show that the diameter of the microspheres is positively correlated with the concentration of chitosan; the diameter of the microspheres increases with increasing chitosan concentration. (From the attached...) Figure 1It can be seen that the microspheres exhibit good sphericity and particle size uniformity, indicating that the prepared microspheres are of good quality. The smaller-diameter microspheres have a darker color, indicating a higher degree of genipin cross-linking. This is because the smaller-diameter microspheres have a larger specific surface area, allowing genipin to diffuse more easily into the interior, thus resulting in a higher degree of cross-linking with chitosan. The compressive strength of the microspheres also increases with increasing chitosan concentration; the compressive strength of microspheres at a concentration of 40 mg / mL is 13.28 MPa, far exceeding the pressure of intravascular blood flow, indicating that the microspheres have clinical application value.
[0026] Example 2 Performance evaluation of microsphere self-developing agent 1. Microsphere preparation process 4.0 g of chitosan was weighed and added to 100 mL of acetic acid solution to prepare a solution with a concentration of 40 mg / mL. 10 mL of the chitosan solution was then added to different masses of iohexol, and the mixture was stirred at room temperature for 30 min (200 rpm) until completely dissolved, yielding a chitosan-iohexol mixed solution. 10 mL of the chitosan-iohexol mixed solution was slowly added to 100 mL of liquid paraffin containing 1.0% SPAN 80, and the mixture was stirred at 1000 rpm for 30 min to form a stable W / O emulsion. 4 mL of genipin solution with a concentration of 1 mg / mL was added dropwise to the emulsion at a rate of 0.5 mL / min, with continuous stirring (1000 rpm). The mixture was reacted at 40℃ in the dark for 4 h. Genipin crosslinked the chitosan with the amino groups on the iohexol to obtain water-containing autoradiographic microspheres. 2. Detection of iodoprolol grafting amount using a UV-Vis spectrophotometer After the reaction was completed, the microspheres were centrifuged and placed in a centrifuge tube with 30 mL of 0.01 M PBS (pH 7.2). The mixture was incubated at 37°C and 180 rpm for 24 h with constant temperature shaking. The eluent was changed every 8 h to ensure complete desorption of free iohexol. The three eluents were combined and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter membrane and brought to a final volume of 100 mL with ultrapure water.
[0027] Using blank PBS as a reference, the absorbance was measured at 245 nm (the characteristic absorption peak of iohexol); iohexol standard solutions with gradients of 8, 15, 25, 40, and 55 μg / mL were prepared, and a standard curve was plotted; the concentration of free iohexol in the eluent was calculated from the standard curve, and thus the content of free iohexol in the microspheres was obtained.
[0028] Calculation of grafted iohexol content: Grafted iohexol content = Total iohexol content - Free iohexol content.
[0029] Iohexol yield (%) = grafted iohexol / iohexol feed amount Cross-linking degree of chitosan amino and iohexol amino (%) = molar fraction of iohexol cross-linked amino / molar fraction of chitosan amino × 100%.
[0030] 3. Investigate the in vitro autoradiography performance of embolization microspheres Calcium alginate-polylysine interpenetrating network microspheres and self-developing microspheres were placed in a 24-well plate and irradiated with X-rays at a height of 1000 µm above the liquid surface using an X-ray machine to observe the development of the microspheres.
[0031] A certain amount of microspheres is added to a sample tube and fixed on a micro-CT sample stage. The parameters are set to scan the sample tube to obtain a two-dimensional projection image sequence of the microspheres, thus obtaining a three-dimensional tomographic image of the microspheres. The CT value of the microspheres is then calculated using the software's built-in program.
[0032] 4. Experimental Results from Figure 2 It can be seen that calcium alginate microspheres lack autoradiographic activity, while those grafted with iohexol exhibit strong radiographic activity, mainly due to the introduction of iodine into the microspheres. Experimental results show that the amount of iohexol grafted onto the microspheres is positively correlated with the iohexol concentration; the amount of iohexol grafted onto the microspheres increases with increasing iohexol concentration. However, the iohexol yield decreases with increasing iohexol concentration. When the iohexol concentration is below 6.0 mg / mL, the yield is only 85.78%, indicating a high grafting efficiency; when the iohexol concentration is above 8.0 mg / mL, the yield is only 85%, indicating a decreased grafting efficiency. Micro-CT measurements of the microspheres' CT values show that the CT values increase with increasing iohexol concentration. When the iohexol concentration is above 6.0 mg / mL, the CT value of the microspheres is greater than 300, indicating that the microspheres have clinical application value. Based on the above experimental results, a concentration of iohexol of 6.0 mg / mL is appropriate, as it ensures that the microspheres have good development properties and a high grafting yield.
[0033] Example 3: Study on the stability of microspheres 1. Microsphere preparation process 4.0 g of chitosan was weighed and added to acetic acid solution to prepare a solution with a concentration of 40 mg / mL. 10 mL of the chitosan solution was added to 60 mg of iohexol, and the mixture was stirred at room temperature for 30 min (200 rpm) until completely dissolved, yielding a chitosan-iohexol mixed solution. 10 mL of the above chitosan-iohexol mixed solution was slowly added to 100 mL of liquid paraffin containing 1.0% (w / v) Span 80, and the mixture was stirred at 1000 rpm for 30 min to form a stable W / O emulsion. 4 mL of genipin solution of different concentrations was added dropwise to the emulsion at a rate of 0.5 mL / min, with continuous stirring (1000 rpm). The mixture was reacted at 40℃ in the dark for 4 h, and the genipin crosslinked the amino groups on the chitosan and iohexol to obtain water-containing self-developing microspheres. After the reaction, the mixture was centrifuged and separated using a solution containing 1.0% (w / v) Tween... The microspheres were washed three times with 80°C deionized water to remove the liquid paraffin from the surface of the microspheres; they were then eluted and dehydrated with a gradient of 30%-50%-70%-95% ethanol, and vacuum dried at room temperature to obtain blue chitosan autoradiographic embolization microspheres.
[0034] 2. Microsphere degradation performance test The dried microspheres were immersed in simulated body fluid (25 mM NaHCO3 solution), and oscillated at a constant temperature of 37°C. The microspheres were periodically removed and dried at 60°C until the weight was constant. The mass loss rate of the microspheres was then measured.
[0035] 3. Experimental Results The experimental results show that the stability of the microspheres increases with the concentration of genipin. When the concentration of genipin is greater than 3.0 mg / mL, the microspheres are stable in the gastrointestinal tract in the simulation test, and the degradation rate is less than 10% after 30 days, indicating that the microspheres can be used in clinical interventional therapy.
[0036] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. A self-developing microsphere composed of chitosan grafted with iodhexol in a one-step process, characterized in that, Using chitosan as the carrier skeleton, iohexol as the self-developing functional component, and genipin, a natural extract, as the cross-linking agent, a one-step method is used to achieve the grafting and cross-linking curing of chitosan and the developer, forming self-developing embolic microspheres with uniform particle size, stable structure, and high mechanical strength.
2. The self-developing microspheres of chitosan grafted with iodhexol using a one-step method according to claim 1, characterized in that, The microspheres have a particle size of 100~1000μm, a CT value ≥300 HU, a compressive strength ≥0.5 MPa, and a mass retention rate ≥90% after being immersed in simulated body fluid for 30 days.
3. A method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process, characterized in that, Includes the following steps: S1. Add chitosan to acetic acid solution and stir at room temperature for 10-30 min until it is completely dissolved; add iodhexol and stir at room temperature for 10-30 min until it is completely dissolved to obtain chitosan-iodhexol mixed solution. S2. The mixed solution obtained in S1 is added to liquid paraffin containing emulsifier and stirred at high speed for 10-30 min to form a stable W / O type emulsion; S3 adds genipin solution dropwise to the emulsion, and stirs the reaction at a constant temperature to achieve grafting and cross-linking curing in one step; After the S4 reaction was completed, the microspheres were centrifuged and washed with deionized water containing 0.5-1.0% (w / v) Tween 80 to remove the liquid paraffin on the microspheres. The microspheres were then eluted and dehydrated using a gradient of 30%-50%-70%-95% ethanol and dried under vacuum at room temperature to obtain blue chitosan autoradiographic embolization microspheres.
4. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S1, iohexol can be replaced with other water-soluble iodine-containing developing agents at a concentration of 2.0~8.0 mg / mL; the chitosan solution concentration is 10~40 mg / mL.
5. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S2, the dispersion medium is liquid paraffin, or one or more mixtures of edible oils; the emulsifier concentration is 0.5~5 wt% (w / v) among the dehydrated sorbitan fatty acid ester surfactants; the volume ratio of the aqueous phase to the oil phase is 1:5~20; and the stirring speed is 1000~5000 rpm.
6. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S3, the concentration of the genipin solution is 1.0~4.0 mg / mL; the ratio of the volume of the genipin solution added to the volume of the chitosan solution is 1:10~20.
7. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S3, the genipin solution drop rate is 0.5~2.0 mL / min; the stirring speed is maintained at 1000~3000 rpm.
8. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S3, the cross-linking reaction time is 3-6 h.
9. The method for preparing self-developing microspheres of chitosan grafted with iodhexol in a one-step process according to claim 3, characterized in that, In step S3, the reaction temperature is 35~45℃.
10. The application of chitosan-grafted iodine-containing autoradiographic microspheres according to claim 1 or 2 in medical imaging tracing.