CT (Computed Tomography) visual embolism microsphere based on iodine-containing nanoparticles as well as preparation method and application of CT visual embolism microsphere
By introducing iodine-containing nanoparticles into interventional embolization microspheres and performing double bond modification, and combining microfluidic control technology to prepare CT visual embolization microspheres, the problem that existing microspheres cannot be accurately positioned in the body is solved, and the TAE therapy is realized CT imaging and biocompatibility improve the reliability of efficacy evaluation.
Patent Information
- Application Number
- CN202411450361.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2025-01-17
AI Technical Summary
Existing interventional embolization microspheres cannot accurately track and locate in vivo, resulting in difficulty in confirming the embolization site and affecting the evaluation of efficacy, especially in CT imaging.
Iodine-containing nanoparticles are prepared by double bond modification and combined with microfluidic control technology. The CT visual embolization microspheres are used to realize the CT imaging function of microspheres by using the high X-ray absorption of nanoparticles, and formed by photocuring hydrogel cross-linking reaction. Uniform microspheres.
The prepared microspheres have good CT imaging effects and biocompatibility, and can accurately display the embolization position in TAE therapy, improving the reliability of efficacy evaluation.
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Figure CN119318730A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a CT visualized embolization microsphere based on iodine-containing nanoparticles and a preparation method and application thereof. Background Art
[0002] Transcatheter arterial embolization (TAE) is a minimally invasive interventional therapy that began in the 1980s and is commonly used to treat tumors such as liver cancer. TAE inserts a catheter into the blood vessel to deliver the embolic agent directly to the artery where the tumor is located, in order to achieve the therapeutic effect of blocking the blood supply to the tumor and promoting tumor necrosis. Arterial embolization can deliver therapeutic drugs such as chemotherapy or radiotherapy at the same time, and is mainly divided into transarterial chemoembolization (TACE) and transarterial radioembolization (TARE). Compared with other treatment strategies, TAE has the advantages of local treatment, reduced side effects, combination with other treatment methods, and a wide range of uses.
[0003] The key to TAE therapy lies in embolic agents, which are currently divided into two categories: liquid embolic agents (iodized oil, anhydrous ethanol, sodium morrhuate, etc.) and solid embolic agents (gelatin sponge, polyvinyl alcohol particles, spring coils, etc.). With the development of materials science, more and more microsphere embolic agents are being used. Compared with other types of embolic agents, embolic microspheres have a smooth surface, are not easy to aggregate, have a uniform particle size, have high targeting to specific tissues and organs, and have good embolic effects. The vast majority of embolic microspheres are prepared using polymers as the basic material. The reason is that polymers are relatively controllable in terms of biocompatibility, lubricity, and biodegradability. According to their degradability, they can be divided into permanent embolic microspheres and biodegradable microspheres, which can adapt to different treatment needs.
[0004] However, the commonly used interventional embolization microspheres have the disadvantage of not being able to develop themselves, and cannot be tracked and located in the body, so it is impossible to accurately determine the distribution of microspheres in blood vessels and the location of embolization. Microsphere visualization can only be assisted by mixing with exogenous contrast agents. However, this indirect visualization method has certain disadvantages. First, in the case of uneven particle distribution, the contrast agent enters the blood vessel farther than the particles. Second, the exogenous contrast agent will quickly diffuse with the blood flow and be quickly cleared from the injection site. The exact location of the embolization microspheres cannot be confirmed after surgery, thus affecting the evaluation of efficacy. Therefore, it is particularly important to give the microspheres their own development function so that they can be visible in blood vessels and tissues.
[0005] At present, the imaging detection methods that can be used in embolization mainly include ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI). Among them, compared with other medical imaging methods, CT imaging has the advantages of better display of calcification and bone lesions, short scanning time, clear images, clear anatomical relationships, and low price. The principle of CT imaging is mainly to detect X-ray photons passing through the body. Therefore, by introducing substances with high X-ray absorption into embolic microspheres, it is expected that the microspheres will have CT imaging functions.
[0006] In summary, in order to identify the location of embolic microspheres during the review of TAE therapy, and then evaluate the embolic area and guide subsequent treatment, it is of great practical significance to develop an embolic microsphere with CT development function. Summary of the invention
[0007] In order to overcome the shortcomings of the above-mentioned prior art, the present invention has developed a method for synthesizing iodine-containing organosilicon nanoparticles, and double-bonded the surface of the nanoparticles to increase the degree of cross-linking with photocurable hydrogels, and then used the above-mentioned iodine-containing nanoparticles in combination with microfluidic technology to prepare embolic microspheres with CT imaging function and good biocompatibility.
[0008] In order to achieve the above object, the technical solution adopted by the present invention is:
[0009] The first aspect of the present invention provides a method for preparing CT-visualized embolization microspheres based on iodine-containing nanoparticles, comprising the following steps:
[0010] S1, dissolving sodium hydroxide and potassium dihydrogen phosphate in water to prepare a phosphate buffer, dissolving CTAB in the obtained buffer, adding (3-iodopropyl)trimethoxysilane, stirring at room temperature for reaction, and dialyzing to remove CTAB to obtain iodine-containing organosilicon nanoparticles;
[0011] S2, adding 3-(methacryloyloxy)propyltrimethoxysilane to the iodine-containing organosilicon nanoparticles of S1, and achieving double bond modification on the surface of the nanoparticles after heating reaction;
[0012] S3. Add methacryloyl gelatin (GelMA) and a photoinitiator to the nanoparticles modified with double bonds on the surface, and use the mixed solution as the dispersed phase of the microfluidic system. Separately, dissolve Span 80 in mineral oil to prepare a mineral oil solution of Span 80, which is used as the mobile phase of the microfluidic system. Then, a cross-shaped microfluidic chip is used to prepare microspheres. The prepared microspheres are then exposed to ultraviolet radiation for cross-linking reaction. After the reaction, a Tween 80 aqueous solution is used as the aqueous phase, and the microspheres obtained by the reaction are allowed to enter the aqueous phase from the mineral oil phase. After washing, hydrogel embolic microspheres are obtained.
[0013] Preferably, in S1, the stirring reaction time at room temperature is 20-30 hours.
[0014] Preferably, in S1, the pH of the phosphate buffer is 7-8.
[0015] Preferably, in S1, the concentration of CTAB in the buffer solution is 70-90 mg / 12 mL, and the usage ratio of CTAB to (3-iodopropyl)trimethoxysilane is 7-9 mg:19-20 μL.
[0016] Preferably, in S2, the heating reaction is carried out at a temperature of 45-60°C and for a time of 40-55 hours.
[0017] Preferably, in S2, the concentration of the iodine-containing organosilicon nanoparticles is 5-50 mg / mL, and the usage ratio of the iodine-containing organosilicon nanoparticles to 3-(methacryloyloxy)propyltrimethoxysilane is 3-7 mL:30-40 μL.
[0018] Preferably, in S3, the photoinitiator comprises lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).
[0019] Preferably, when a cross-shaped microfluidic chip is used to prepare microspheres in S3, the flow rate of the dispersed phase is set to 2-5 μL / min, and the flow rate of the mobile phase is set to 7-13 μL / min.
[0020] Preferably, in S3, the ultraviolet irradiation time is 3-7 min, the wavelength is 405 nm, and the irradiation intensity is 2-30 mW / cm 2 .
[0021] Preferably, in S3, the mass concentration of the mineral oil solution in the Span 80 is 8-15%.
[0022] Preferably, in S3, based on the nanoparticles before modification, the concentration of the nanoparticles is not less than 10 mg / mL, and the usage ratio of the nanoparticles to GelMA and the photoinitiator is 1-3 mL: 70-120 mg: 4-7 mg.
[0023] The second aspect of the present invention provides CT visualized embolization microspheres based on iodine-containing nanoparticles prepared by the preparation method described in the first aspect.
[0024] The third aspect of the present invention provides the use of the CT-visualized embolic microspheres based on iodine-containing nanoparticles described in the second aspect in the preparation of TAE embolic agents.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] The invention discloses a method for preparing CT visualized embolization microspheres based on iodine-containing nanoparticles. First, (3-iodopropyl) trimethoxysilane is hydrolyzed and condensed in a phosphate buffer solution under the action of a surfactant, hexadecyl trimethylammonium bromide (CTAB), to form nanoparticles. After removing CTAB by dialysis, 3-(methacryloyloxy) propyl trimethoxysilane is allowed to continue to undergo hydrolysis and condensation reaction on the surface of the nanoparticles, thereby introducing active double bonds on the surface of the nanoparticles to prepare iodine-containing organosilicon nanoparticles. Then, methacryloyl gelatin (GelMA) and a photoinitiator are added to the iodine-containing organosilicon nanoparticles to form a photocurable cross-linked aqueous phase, which is used as a dispersed phase in a microfluidic system. In addition, a mineral oil solution of Span 80 is prepared as a mobile phase in the microfluidic system, and then the preparation of microspheres is realized by a cross-shaped microfluidic chip. The embolization microspheres prepared by the method of the invention have a relatively uniform size distribution, good CT imaging effect, and good biocompatibility, and have the potential to be applied to TAE therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a technical roadmap for the preparation and modification of iodine-containing organosilicon nanoparticles;
[0028] Figure 2 Schematic diagram of the preparation of CT-visualized embolic microspheres based on iodine-containing nanoparticles;
[0029] Figure 3 The hydrated particle size and Zeta potential of iodine-containing nanoparticles before (a) and after (b) modification;
[0030] Figure 4 Transmission electron microscopy of iodine-containing nanoparticles before and after modification;
[0031] Figure 5 Infrared spectra of iodine-containing nanoparticles before and after double bond modification;
[0032] Figure 6 This is an optical microscope photo of embolic microspheres;
[0033] Figure 7 The images of embolic microspheres doped with iodine-containing nanoparticles at different concentrations under a medical CT scanner;
[0034] Figure 8 The figure shows the cytotoxicity evaluation of embolic microspheres doped with iodine-containing nanoparticles at different concentrations (MRC-5 cell line, 24 h). DETAILED DESCRIPTION
[0035] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.
[0037] Example: A method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles
[0038] (1) Synthesis of iodine-containing organosilicon nanoparticles ( Figure 1 ) and characterization
[0039] 580mg sodium hydroxide and 3400mg potassium dihydrogen phosphate were dissolved in 500mL deionized water to form a buffer solution with a pH of 7.4. 120mL of the buffer solution was placed in a round-bottom flask, and 880mg CTAB was added under stirring. After the CTAB was completely dissolved, 1920μL (3-iodopropyl) trimethoxysilane was added dropwise, and the mixture was stirred at room temperature (speed 700rpm) for 24h. The reaction solution was placed in a dialysis bag of 8000-14000Da, and the CTAB was removed by dialysis with a mixed solution of ethanol and water in a volume ratio of 1:13 and deionized water for more than 5 days to obtain iodine-containing organosilicon nanoparticles.
[0040] The particle size and Zeta potential of the prepared nanoparticles were characterized by a nanoparticle size and Zeta potential analyzer, and the particle size of the nanoparticles was uniform (e.g. Figure 3 a), the average particle size is 45.66nm, and the Zeta potential is 27.13mV. Further analysis by transmission electron microscopy ( Figure 4 ) showed that the nanoparticles were spherical with a particle size of 29-42nm, which was similar to the results of dynamic light scattering (DLS) particle size measurement.
[0041] (2) Modification and characterization of double bonds on the surface of nanoparticles
[0042] In a 50 mL reaction tube, add 5 mL of the dialyzed purified iodine-containing nanoparticle aqueous solution (the mass concentration of which was determined by freeze-drying to be approximately 10 mg / mL), then add 35 μL of 3-(methacryloyloxy)propyltrimethoxysilane while stirring, and heat to 50°C for reaction for 48 h.
[0043] After the reaction was terminated, the particle size and Zeta potential were characterized using a nanoparticle size and Zeta potential analyzer, and the particle size of the nanoparticles was uniform (e.g. Figure 3b), the average particle size was 53.86 nm, which was increased compared to that before modification, and the Zeta potential was 27.13 mV. At the same time, in order to verify that the surface of the nanoparticles was modified with double bonds, part of the reaction solution was put into a 8000-14000Da dialysis bag, and dialyzed for more than 3 days using a mixed solution of ethanol and water in a volume ratio of 1:13 to remove unreacted 3-(methacryloyloxy)propyltrimethoxysilane, and the dialyzed reaction solution was freeze-dried into powder. Another part of the nanoparticle emulsion before modification was also freeze-dried. The nanoparticle powders before and after modification were then characterized by infrared absorption spectra using the tabletting method. It was known from the infrared spectrum (such as Figure 5 ), compared with the nanoparticles before modification, the modified nanoparticles have a wavelength of 1600-1800cm -1 The infrared absorption peak of the carbon-carbon double bond appeared at the nanoparticles, proving that the carbon-carbon double bond was successfully modified.
[0044] (3) Preparation of hydrogel embolic microspheres Figure 2 ) and characterization
[0045] According to the formula C1V1=C2V2 (C1 and C2 are the concentrations before and after concentration, respectively, and V1 and V2 are the volumes before and after concentration, respectively), the nanoparticles modified with double bonds on the surface were concentrated to different mass concentrations (18 mg / mL, 25 mg / mL, 33 mg / mL, where the concentration of the nanoparticles was calculated based on the concentration before modification) by rotary evaporation, and then 100 mg of methacryloyl gelatin (GelMA, double bond modification degree of about 60%, purchased from Aladdin) and 5 mg of photoinitiator were added to 1 mL of the concentrated solution. The agent phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) was added, and the mixed solution was used as the dispersed phase of the microfluidic system; 10g of Span 80 was weighed and dissolved in 90g of mineral oil to prepare a mineral oil solution of Span 80 with a mass fraction of 10%, which was used as the mobile phase of the microfluidic system. Then, a common cross-shaped microfluidic chip (purchased from Guangzhou National Laboratory) was used to prepare microspheres, where the flow rate of the dispersed phase was set to 3μL / min and the flow rate of the mobile phase was set to 10μL / min. The obtained microspheres were then collected in a centrifuge tube and irradiated under ultraviolet light for 4 minutes for cross-linking reaction (wavelength of 405nm, irradiation intensity of 25mW / cm 2 Then, 20 g of Tween 80 was weighed and dissolved in 980 mL of pure water to prepare a Tween 80 aqueous solution with a mass concentration of 2%. The Tween 80 aqueous solution was mixed with the microsphere & mineral oil mixed system after the reaction at a volume ratio of 100:1. After centrifugation at 2000 rpm for 6 min to allow the microspheres to enter the water phase from the mineral oil phase, deionized water was used for centrifugal washing for more than 5 times to obtain three types of hydrogel embolic microspheres containing nanoparticles of different concentrations and with a size range of 60-100 μm (the optical microscope observation results are shown in FIG. Figure 6 and freeze-dried for storage.
[0046] In addition, GelMA microspheres without nanoparticles were prepared by a similar method as a control, except that the nanoparticle concentrate was not used in the preparation process.
[0047] Experimental example: Performance test of CT-visualized embolization microspheres based on iodine-containing nanoparticles
[0048] 1. Characterization of in vitro CT imaging performance:
[0049] Weigh 100 mg of each of the three freeze-dried microspheres containing different concentrations of nanoparticles in the example in a centrifuge tube, add 3 mL of PBS solution to allow them to fully absorb water and swell, and filter them to remove excess water, and then transfer them to a 2 mL centrifuge tube. At the same time, GelMA microspheres without nanoparticles were prepared in the same way and used as blank control groups for the test together with PBS solution. After sample preparation, the samples were scanned using a CT scanner, and the generated data files were processed and analyzed using RadiAnt DICOM Viewer. The results are shown in Figure 2. Figure 7 As shown, compared with the blank microsphere group and the PBS solution group, the three experimental groups all showed good CT imaging effects, and the CT values gradually increased with the increase in the concentration of iodine-containing nanoparticles.
[0050] 2. Cytotoxicity characterization:
[0051] The MRC-5 cell line was used to characterize the cytotoxicity of three freeze-dried microspheres containing different concentrations of nanoparticles in the example by the CCK-8 method. About 5000 cells were plated in each well of a 96-well plate, and the cells were incubated with three different concentrations of microspheres for 24 hours. After removing the supernatant, 10 μL of CCK-8 reagent and 90 μL of DMEM culture medium were added to each well and incubated for another 1.5 hours (the concentrations of the microspheres in the culture medium were 100 ug / mL, 500 ug / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL, respectively). Finally, the relative viability of the cells was determined by an ELISA instrument (calculated by the absorbance at 450 nm). The test results are as follows: Figure 8 As shown, it can be seen that each experimental group showed good biocompatibility, and the cell viability was above 80% within the set concentration range.
[0052] In summary, the present invention uses (3-iodopropyl)trimethoxysilane and 3-(methacryloyloxy)propyltrimethoxysilane to prepare nanoparticles with surface modified active double bonds, thereby realizing the synthesis of iodine-containing organosilicon nanoparticles. At the same time, after double bond modification on the surface of the nanoparticles, the embolic microspheres prepared in combination with microfluidic technology have both CT imaging function and good biocompatibility, and have the potential to be applied to TAE therapy.
[0053] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.
Claims
1. A method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles, characterized in that: The following steps are involved: S1, dissolving sodium hydroxide and potassium dihydrogen phosphate in water to prepare a phosphate buffer, dissolving CTAB in the obtained buffer, adding (3-iodopropyl)trimethoxysilane, stirring at room temperature for reaction, and dialyzing to remove CTAB to obtain iodine-containing organosilicon nanoparticles; S2, adding 3-(methacryloyloxy)propyltrimethoxysilane to the iodine-containing organosilicon nanoparticles of S1, and achieving double bond modification on the surface of the nanoparticles after heating reaction; S3. Add methacryloyl gelatin (GelMA) and a photoinitiator to the nanoparticles modified with double bonds on the surface, and use the mixed solution as the dispersed phase of the microfluidic system. Separately, dissolve Span 80 in mineral oil to prepare a mineral oil solution of Span 80, which is used as the mobile phase of the microfluidic system. Then, a cross-shaped microfluidic chip is used to prepare microspheres. The prepared microspheres are then exposed to ultraviolet radiation for cross-linking reaction. After the reaction, a Tween 80 aqueous solution is used as the aqueous phase, and the microspheres obtained by the reaction are allowed to enter the aqueous phase from the mineral oil phase. After washing, hydrogel embolic microspheres are obtained.
2. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S1, the stirring reaction time at room temperature is 20-30 hours.
3. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S1, the pH of the phosphate buffer is 7-8.
4. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S2, the heating reaction is carried out at a temperature of 45-60°C and for a time of 40-55 hours.
5. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S3, the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).
6. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: When the cross-shaped microfluidic chip was used to prepare microspheres in S3, the flow rate of the dispersed phase was set to 2-5 μL / min, and the flow rate of the mobile phase was set to 7-13 μL / min.
7. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S3, the UV irradiation time is 3-7min, the wavelength is 405nm, and the irradiation intensity is 2-30mW / cm 2 .
8. The method for preparing CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 1, characterized in that: In S3, the mass concentration of the mineral oil solution of Span 80 is 8-15%.
9. CT-visualized embolization microspheres based on iodine-containing nanoparticles prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the CT-visualized embolic microspheres based on iodine-containing nanoparticles according to claim 9 in the preparation of TAE embolic agents.