A 3D printing light-blocking agent based on flavonoid nanoparticles and its preparation method and application

Abstract

The present invention proposes a three-dimensional printing light-blocking agent based on flavonoid nanoparticles, and a preparation method and application thereof, which belongs to the field of 3D printing technology. The raw materials of the three-dimensional printing light-blocking agent based on flavonoid nanoparticles of the present invention include a flavonoid nanoparticle solution. The present invention greatly improves the water solubility of flavonoid compounds through intermolecular self-assembly, and expands the application space of flavonoid compounds in aqueous phase systems. The biological scaffold prepared by the light-blocking agent has good printing accuracy and promotes the survival of internal cells while releasing flavonoids to exert antioxidant effects. The preparation process of the present invention is highly controllable, and a variety of processing and manufacturing methods can be used to obtain scaffold materials with the desired structure and desired biochemical properties, which is suitable for the preparation of repair materials with customized size and morphology.

Description

Technical Field

[0001] The present invention belongs to the technical field of 3D printing, and in particular relates to a 3D printing light-blocking agent based on flavonoid nanoparticles, and a preparation method and application thereof. Background Art

[0002] As an advanced material processing and preparation method, the main feature of three-dimensional (3D) printing technology lies in its "additive" preparation method. In this process, complex 3D objects are first designed as needed through computer-aided digital design, and then objects with complex internal structures are directly generated in three dimensions accurately and quickly according to the design. The photopolymerization-based biological 3D printing method can load cells into photopolymerized hydrogels, reproduce the complexity of the three-dimensional environment surrounding cells in the body to the greatest extent, and enable cells to perform more sound biological functions in vitro. At present, biological 3D printing has played an important role in biomedical engineering and is widely used in the processing and preparation of tissue engineering scaffolds. It is also expected to become a basic tool for biomedical research.

[0003] Among various light-based 3D printing technologies, they can be roughly divided into two categories: line-to-line stacking printing represented by traditional extrusion printing, which is cured after extrusion; the other method is surface-to-surface stacking printing represented by SLA and DLP printing, which is cured layer by layer through light projection. Since the curing of photosensitive ink is directly related to the distribution of light on a two-dimensional plane during the printing process of SLA and DLP, it can improve the processing accuracy by modulating the light; at the same time, the "surface-to-surface stacking" printing method also determines its faster preparation speed. Therefore, SLA and DLP have become the most widely used 3D printing technologies. In addition, light-based printing technology has good compatibility with various biocompatible photosensitive ink systems, such as gelatin methacryloyl (GELMA), methacrylate hyaluronic acid (HAMA) and polyethylene glycol diacrylate (PEGDA), which can maintain the activity of loaded cells in biological 3D printing. At the same time, the bio-ink required for light-based printing has a low viscosity, which makes it easier and more efficient to add various active substances, including various drugs and growth factors, which further expands its application in the field of biomedical engineering.

[0004] However, due to the inherent limitations of the optical properties of inks, light-based printing does not always achieve the expected accuracy. On the one hand, due to the inconsistency of the refractive index between the curing gel and the printing ink, as well as between the loaded cells and the printing ink, diffuse scattering occurs between the interfaces of the two, causing the light to deviate from the preset track and reduce the printing accuracy; on the other hand, the penetration depth of the curing light in light-based printing is difficult to control, causing the light to penetrate the printed structure, thereby causing distortion of the printed structure. These problems limit the application of light-based printing in the field of bioengineering and medicine.

[0005] In order to eliminate the deterioration of the printed structure caused by unnecessary polymerization in light-based printing, a common method is to add a light absorber to the printing ink, which has the ability to absorb irradiated light, and dissipates most of the energy in the form of heat by absorbing the irradiated light, so that the cross-linking reaction caused by irradiation is suppressed within a certain area, thereby improving the printing accuracy. At present, most light absorbers are food dyes, such as tartrate yellow (Tar), reactive orange 16 (RO16), acid red dyes and anthocyanins, etc., and the accuracy of the printed structure is improved by adding these light absorbers. However, with the deepening of research, many results show that these widely used light absorbers have limitations in performance, and they still cannot suppress the light scattering caused by suspended cells. At the same time, the above-mentioned light absorbers dissipate most of the energy in the light when they work, slowing down the printing speed. In addition, most light absorbers are cytotoxic, and currently only tartrate yellow light absorber has been proven to be non-toxic, so it is often not selected when carrying out cell-based printing.

[0006] In addition, in order to improve the accuracy of light-based printing, many methods that are not based on light absorbers have been reported in the prior art. These methods are based on optimizing the existing printing process, using artificial intelligence and other methods to dynamically adjust the horizontal distribution of light intensity to minimize the impact of scattering; or adjusting the refractive index of the ink so that it can match the refractive index of the cells to reduce the scattering caused by the cells. However, these methods do not truly eliminate the refraction and over-penetration of light, and whether they can play a role in the printing of larger models remains to be confirmed.

[0007] In summary, in order to achieve more efficient light-based printing, a new cell-compatible strategy is needed, which should have higher printing accuracy, faster processing speed and better operability, so as to achieve a more efficient light-based bioprinting manufacturing method, which is obviously of great significance for the construction of bionic models with more complex structures and scales closer to tissues and even organs. Summary of the invention

[0008] In order to solve the above technical problems, the present invention proposes a 3D printing light-blocking agent based on flavonoid nanoparticles, and a preparation method and application thereof. The prepared light-blocking agent has good cell compatibility and biological activity, and its water solubility is improved, so that it can play a role in improving the precision in the preparation of photocurable scaffold materials.

[0009] To achieve the above-mentioned purpose, the present invention provides a three-dimensional printing light-blocking agent based on flavonoid nanoparticles, wherein the raw materials include a flavonoid nanoparticle solution, and the flavonoid compounds used in the preparation process of the flavonoid nanoparticle solution include one or more of rutin, rutin, epigallocatechin gallate, apigenin, genistein, hesperetin, quercetin, daidzein and naringenin.

[0010] Furthermore, the method for preparing the flavonoid nanoparticle solution comprises the following steps:

[0011] The flavonoid compound is mixed with a buffer solution, and the pH is adjusted to obtain the flavonoid nanoparticle solution.

[0012] Furthermore, the dosage ratio of the flavonoid compound to the buffer solution is (0.025-0.3) g: 3 mL.

[0013] Furthermore, the pH is adjusted to 7-9.

[0014] Furthermore, the buffer solution is at least one of PBS, HBSS, Tris-HCl and sodium carbonate buffer or a mixture of any proportions of several of them.

[0015] Furthermore, the raw materials also include a water-soluble photoinitiator, and the usage ratio of the flavonoid nanoparticle solution to the water-soluble photoinitiator is 10 mL: 0.025 g.

[0016] As the main component of various traditional Chinese medicines and foods, flavonoids are widely available and have various beneficial biological functions. They are prepared into flavonoid-based nanoparticles with uniform particle size and excellent water solubility through pH-adjusted self-assembly technology, thereby improving the water solubility of flavonoids. This nanoparticle has good absorption capacity at the printing wavelength and can play the role of a light absorber; at the same time, it retains the free radical scavenging ability common to flavonoids, and can further improve the printing accuracy by removing excess chain free radicals; in addition, flavonoids are harmless ingredients in various traditional Chinese medicines and health foods, and the nanoparticles prepared with them also have good cell compatibility and can maintain cell survival, which is conducive to their application in bioprinting and solves the problem of cytotoxicity of most light absorbers.

[0017] The present invention also proposes a method for preparing the 3D printing light-blocking agent based on flavonoid nanoparticles, comprising the following steps:

[0018] The flavonoid nanoparticle solution is mixed and diluted with a buffer solution, and then a water-soluble photoinitiator is added, and after mixing, the three-dimensional printing light-blocking agent based on flavonoid nanoparticles is obtained.

[0019] Furthermore, the flavonoid nanoparticle solution is mixed with a buffer solution and diluted to a concentration of 0.001-1.0 g / mL.

[0020] The present invention also proposes the use of the flavonoid nanoparticle-based three-dimensional printing light-blocking agent in the preparation of a photocurable bracket material, wherein the flavonoid nanoparticle-based three-dimensional printing light-blocking agent is mixed with a water-soluble photocurable resin to form a photosensitive ink solution, and the photosensitive ink solution is fixed by photocuring to obtain a photocurable bracket material.

[0021] Furthermore, the water-soluble photocurable resin includes at least one of PEGDA, GELMA, F127DA, HAMA, ALGMA, SILMA, PLMA, GELNB and HANB, or a mixture of any proportions of the above.

[0022] Furthermore, the photocuring shaping method includes ultraviolet light curing, photocuring in-situ 3D printing, SLA 3D printing, DLP 3D printing and two-photon lithography 3D printing.

[0023] Furthermore, when the photocuring method uses ultraviolet light curing, the obtained photosensitive ink solution is added to the mold, and the degree of gelation is regulated by parameters such as irradiation light intensity, irradiation time, and irradiation angle, and the photocurable bracket material of the desired shape is obtained after demolding.

[0024] Furthermore, when the photocuring shaping method adopts photocuring in-situ 3D printing, the obtained photosensitive ink solution is loaded into a barrel, the barrel is installed on a 3D printer, and the diameter of the extruded gel and the degree of gelation are regulated by adjusting the three-dimensional (x, y, z axis) movement trajectory of the barrel, the speed of the barrel advancing piston, the thickness of the needle, the speed and lateral movement speed of the receiving rod, the intensity of the irradiated light and the irradiation time and other parameters. Thus, the photocurable scaffold material of the desired shape is obtained.

[0025] Furthermore, when the photocuring shaping method adopts SLA 3D printing or DLP 3D printing, the obtained photosensitive ink solution is loaded into a material tank, and the deposition platform is installed on the 3D printer. The gel is well formed by adjusting parameters such as the slice layer height, single-layer irradiation light intensity and irradiation time, peeling speed and scraping speed, thereby obtaining a photocurable scaffold material of the desired shape.

[0026] Furthermore, when the photocuring shaping method adopts two-photon lithography 3D printing, the obtained photosensitive ink solution is loaded into a material tank, and the deposition platform is installed on the 3D printer. By adjusting parameters such as laser power, laser focus offset speed and calibration platform horizontal position, the gel is precisely formed at a specified position, thereby obtaining a photocurable scaffold material of a desired shape.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] (1) The 3D printing light-blocking agent based on flavonoid nanoparticles of the present invention is derived from flavonoid compounds, retains its favorable physical and chemical properties, greatly improves the water solubility of flavonoid compounds through intermolecular self-assembly, and expands the application space of flavonoid compounds in aqueous phase systems.

[0029] (2) Compared with the edible pigment light absorber, the 3D printing light blocker based on flavonoid nanoparticles of the present invention has a similar light absorption capacity but a weaker dissipation effect on light energy, thereby improving the energy utilization efficiency of the light reaction and accelerating the printing speed; at the same time, it has the free radical scavenging ability that traditional light absorbers do not have, and can block malicious polymerization reactions by reacting with chain free radicals, further improving printing accuracy.

[0030] (3) Compared with the photocurable scaffold material prepared by using the flavonoid nanoparticle-based three-dimensional printing light-blocking agent of the present invention, the biocompatibility of the photocurable scaffold material is improved due to the addition of flavonoid nanoparticles; in addition, the flavonoids can be released to exert their beneficial physiological functions, such as anti-inflammation, anti-oxidation, anti-apoptosis and regulation of mitochondrial energy metabolism, further improving the repair effect.

[0031] (4) The preparation technology of the present invention is highly controllable, and a variety of processing and manufacturing methods can be used to obtain scaffold materials with desired structures and desired biochemical properties, which is suitable for the preparation of repair materials with customized size and morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0033] Figure 1 The following are the appearance photos (A) and transmission electron microscope (TEM) photos (B) of different flavonoid nanoparticle solutions in Examples 1-8;

[0034] Figure 2 Absorption spectra of different flavonoid nanoparticle solutions in Examples 1-8 (A) and absorbance at wavelengths of 405 nm (B) and 365 nm (C);

[0035] Figure 3 The free radical scavenging ability test results of different flavonoid nanoparticle solutions in Examples 1-8, wherein A is the scavenging ability test result of DPPH, and B is the scavenging ability test result of ABTS;

[0036] Figure 4 The results of the photothermal dissipation capacity measurement of the flavonoid nanoparticle solution in Examples 1-8 at working wavelengths of 405 nm (A) and 365 nm (B);

[0037] Figure 5 The printing resolution of the bio-ink using different flavonoid nanoparticles in Application Examples 1-8 at 405 nm (A) and 365 nm (B);

[0038] Figure 6 The cell compatibility of the hydrogel scaffolds prepared in Application Examples 1-8 and the Blank group on the 1st, 3rd and 5th days;

[0039] Figure 7 The results of the antioxidant assay of the hydrogel scaffolds in Application Examples 1-8 and the Blank group, where A is a representative image after ROS probe labeling, and B is the intracellular ROS concentration in each group;

[0040] Figure 8 The actual printing performance of flavonoid nanoparticles in complex bioprinting scenarios. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The sources of the raw materials used in the embodiments of the present invention are as follows:

[0047] Rutin (RUT): Shanghai MacLean Biochemical Technology Co., Ltd.;

[0048] Epigallocatechingallate (EGCG): Shanghai MacLean Biochemical Technology Co., Ltd.;

[0049] Apigenin (API): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0050] Genistein (GEN): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0051] Hesperitin (HES): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0052] Quercetin (QUE): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0053] Daidzein (DAI): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0054] Naringenin (NAR): Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0055] Phosphate buffered saline (PBS): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0056] HBSS-Hank's balanced salt solution (HBSS): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0057] Tris-HCl buffer: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0058] Carbonate buffer: Shanghai Jingkang Biological Co., Ltd.;

[0059] Sodium hydroxide (NaOH): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0060] 37% hydrochloric acid (HCl): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0061] Anhydrous calcium chloride (CaCl2): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0062] Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP): Shanghai TCI Chemical Industry Development Co., Ltd.;

[0063] Polyethylene glycol diacrylate (PEGDA): Sigma aldrich (St. Louis, MO, USA);

[0064] Polyether pluronic diacrylate (F127DA): Sigma aldrich (St. Louis, MO, USA);

[0065] Sodium methacrylate alginate (ALGMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.;

[0066] Methacrylated silk fibroin (SILMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.;

[0067] Methacrylated hyaluronic acid (HAMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.;

[0068] Methacrylated gelatin (GELMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.;

[0069] Methacrylated lysine (PLMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.;

[0070] Norbornene-modified gelatin (GELNB): Sigma aldrich (St. Louis, MO, USA);

[0071] Norbornene-modified hyaluronic acid (HANB): Sigma aldrich (St. Louis, MO, USA).

[0072] In the embodiments of the present invention, precise pH test paper and a pH meter are used to accurately measure pH.

[0073] The technical solution of the present invention is further illustrated by the following embodiments.

[0074] Example 1

[0075] A method for preparing a rutin nanoparticle solution comprises the following steps:

[0076] 0.025 g of rutin powder was dissolved in 2 mL of 0.1 M NaOH solution, and vortexed until completely dissolved. Then, 3 mL of 0.01 M phosphate buffer was slowly dripped in while stirring. 13 μL of concentrated hydrochloric acid (i.e., 37% hydrochloric acid) was added while vortexing, and the pH was adjusted to 7.4 to obtain a hydrogel presolution. The prepared hydrogel presolution was filtered using a 0.22 μm syringe filter in an ultra-clean bench to obtain a rutin nanoparticle solution (RUT).

[0077] Example 2

[0078] Same as Example 1, except that the mass of rutin is replaced by quercetin (QUE).

[0079] Example 3

[0080] Same as Example 1, except that rutin is replaced by apigenin (API).

[0081] Example 4

[0082] Same as Example 1, except that the mass of rutin is replaced by genistein (GEN).

[0083] Example 5

[0084] Same as Example 1, except that the mass of rutin is replaced by hesperidin (HES).

[0085] Example 6

[0086] Same as Example 1, except that rutin is replaced by daidzein (DAI).

[0087] Example 7

[0088] Same as Example 1, except that the mass of rutin is replaced by naringenin (NAR).

[0089] Example 8

[0090] Same as Example 1, except that the mass of rutin is replaced by epigallocatechin gallate (EGCG).

[0091] Performance test of different flavonoid nanoparticle solutions

[0092] Figure 1 The following are the appearance photos (A) and transmission electron microscope (TEM) photos (B) of different flavonoid nanoparticle solutions in Examples 1-8. Figure 1 It can be seen that each group of flavonoid nanoparticles is evenly dispersed in water and has a stable microscopic morphology that is close to a spherical shape.

[0093] Figure 2 The absorption spectra of different flavonoid nanoparticle solutions in Examples 1-8 (A) and the absorbance at wavelengths of 405nm (B) and 365nm (C). Figure 2 It can be seen that all groups of flavonoid nanoparticles have strong absorption at 365nm; except for EGCG and GEN, all other groups have strong absorption at 405nm. Among them, 365nm and 405nm are commonly used working wavelengths for 3D printers. This shows that flavonoid nanoparticles can play a role in improving precision through light absorption.

[0094] Figure 3 The free radical scavenging ability test results of different flavonoid nanoparticle solutions in Examples 1-8, wherein A is the scavenging ability test result of DPPH, and B is the scavenging ability test result of ABTS. Figure 3 It can be seen that each group of flavonoid nanoparticles has good free radical scavenging ability, especially when scavenging ABTS free radicals in water, which shows that the flavonoid nanoparticles prepared by this technology have good water solubility and can therefore play a role in aqueous biological 3D printing.

[0095] Figure 4 The photothermal dissipation capacity of the flavonoid nanoparticle solution in Examples 1-8 at working wavelengths of 405 nm (A) and 365 nm (B) is measured. Figure 4 It can be seen that the photothermal dissipation capacity of each group of flavonoid nanoparticles at the working wavelength is positively correlated with its absorption at that wavelength, further proving its mechanism of improving accuracy based on light absorption.

[0096] Example 9

[0097] The preparation of a 3D printing light-blocking agent based on a flavonoid nanoparticle solution comprises the following steps:

[0098] The flavonoid nanoparticle solutions prepared in Examples 1-8 were diluted with 0.01 M phosphate buffer to a concentration of 0.001 g / mL, 10 mL of the above solution was added with 0.025 g of a water-soluble photoinitiator Lap, and vortexed at 37° C. until completely dissolved to obtain a three-dimensional printing light-blocking agent based on the flavonoid nanoparticle solution.

[0099] Application Example 1

[0100] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of epigallocatechin gallate nanoparticles includes the following steps:

[0101] 0.9 mL of PEGDA and 0.5 g of GELNB powder freeze-dried block were added to 10 mL of the 3D printing light-blocking agent based on the epigallocatechin gallate nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution;

[0102] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slices, and print. After printing is completed, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% EGCG) based on the photoinhibition of epigallocatechin gallate nanoparticles.

[0103] Application Example 2

[0104] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of apigenin nanoparticles includes the following steps:

[0105] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the 3D printing light-blocking agent based on the apigenin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution;

[0106] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% API) based on the photoinhibition of apigenin nanoparticles.

[0107] Application Example 3

[0108] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of genistein nanoparticles includes the following steps:

[0109] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the 3D printing light-blocking agent based on the genistein nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in an ultra-clean bench to obtain a photosensitive ink solution;

[0110] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% GEN) based on the light inhibition of genistein nanoparticles.

[0111] Application Example 4

[0112] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of daidzein nanoparticles includes the following steps:

[0113] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the 3D printing light-blocking agent based on the soy aglycone nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution;

[0114] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% DAI) based on the photoinhibition of soybean aglycone nanoparticles.

[0115] Application Example 5

[0116] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of quercetin nanoparticles includes the following steps:

[0117] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the quercetin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution;

[0118] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product based on the photoinhibition of quercetin nanoparticles (0.1% QUE).

[0119] Application Example 6

[0120] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of hesperidin nanoparticles includes the following steps:

[0121] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the three-dimensional printing light-blocking agent based on the hesperidin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in an ultra-clean bench to obtain a photosensitive ink solution;

[0122] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% HES) based on the light inhibition of hesperidin nanoparticles.

[0123] Application Example 7

[0124] The preparation of DLP-printed hydrogel scaffolds based on the photoinhibition of naringenin nanoparticles includes the following steps:

[0125] 0.9 mL of PEGDA and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the naringenin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution;

[0126] Adjust the printer parameters: the printing light intensity is set to 15mv / cm2, the layer height is set to 100μm, the exposure time is 6s, the number of base layers is 5, the base exposure time is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. Add the obtained photosensitive ink solution to the material tank, import the model slice, and print. After printing, peel the model from the deposition platform to prepare a DLP printed hydrogel scaffold product (0.1% NAR) based on the light inhibition of naringenin nanoparticles.

[0127] Application Example 8

[0128] The preparation of a hydrogel cast scaffold loaded with rutin nanoparticles comprises the following steps:

[0129] 0.2 g of ALGMA was added to 10 mL of the 3D printing light-blocking agent based on the rutin nanoparticle solution prepared in Example 9, and the mixture was vortexed at 37° C. until completely dissolved to obtain a photosensitive ink solution;

[0130] The prepared photosensitive ink solution was poured into the prepared PDMS mold, and the bubbles were removed by a circulating vacuum water pump for 30 minutes, the vacuum degree was maintained at 0.1 MPa, and the excess bubbles were removed by a 1 mL pipette. After that, it was irradiated with a 405 nm UV curing lamp for 5 minutes, and demolded after complete curing. After that, it was immersed in a 5 mg / mL calcium chloride solution for crosslinking for 5 minutes to obtain a hydrogel casting scaffold product loaded with rutin nanoparticles (0.1% RUT).

[0131] Application Example 9

[0132] The preparation of SLA-printed hydrogel scaffolds based on the photoinhibition of hesperidin nanoparticles includes the following steps:

[0133] 0.3 g of HANB and 0.5 g of GELNB freeze-dried cake were added to 10 mL of the three-dimensional printing light-blocking agent based on the hesperidin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in an ultra-clean bench to obtain a photosensitive ink solution;

[0134] The printer parameters were adjusted: 50 μm fiber UV laser beam, 25 kHz UV emission, 25 μJ output intensity, and 10 mm / s printing speed. The obtained photosensitive ink solution was added to the material tank, and the CAD model slices were imported for printing. After printing, the model was peeled off from the deposition platform to prepare an SLA printed hydrogel scaffold product based on the photoinhibition of hesperidin nanoparticles.

[0135] Application Example 10

[0136] Preparation of DLP printed hydrogel scaffolds loaded with PC12 cells based on the photoinhibitory properties of epigallocatechin gallate nanoparticles, comprising the following steps:

[0137] 0.5 g of GelMA freeze-dried block and 0.05 g of PLMA freeze-dried block were added to 10 mL of the 3D printing light-blocking agent based on the epigallocatechin gallate nanoparticle solution prepared in Example 9, vortexed at 37°C until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench. Then, 3*10 7 PC12 cells to obtain a photosensitive bio-ink solution;

[0138] Adjust the printer parameters: set the printing intensity to 15mv / cm 2 , the layer height is set to 100μm, the exposure time is 17s, the number of base layers is 5, the base exposure time is 22s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. The obtained photosensitive ink solution is added to the material tank, imported into the model slice, and printed. After printing, the model is peeled off from the deposition platform to prepare a DLP printed hydrogel scaffold product containing PC12 cells based on the photoinhibition of rutin nanoparticles.

[0139] Application Example 11

[0140] Preparation of a multi-material DLP-printed hydrogel scaffold loaded with photoinhibitory quercetin nanoparticles, comprising the following steps:

[0141] 0.5 g of SILMA freeze-dried block and 0.25 g of HANB were added to 10 mL of the quercetin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution A;

[0142] 2.25 mL of PEGDA was added to 10 mL of the quercetin nanoparticle solution prepared in Example 9, vortexed at 37° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution B;

[0143] 0.9 mL of PEGDA and 0.5 g of F127DA were added to 10 mL of the quercetin nanoparticle solution prepared in Example 9, vortexed at 4° C. until completely dissolved, and filtered using a 0.22 μm syringe filter in a clean bench to obtain a photosensitive ink solution C;

[0144] The prepared photosensitive ink solutions A, B, and C were filtered using a 0.22 μm syringe filter in a clean bench.

[0145] Add photosensitive ink solutions A, B, and C to different slots of the multi-material printer and name them as slots A, B, and C. Import the model slices and associate the corresponding parts with slots A, B, and C according to the design. Set the corresponding parameters of the slots, and the light intensity is 15mv / cm 2 , the layer height is set to 100μm, the exposure time of slot A is 7s, the number of base layers is 3, the exposure time of base layer is 10s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s; the exposure time of slot B is 4.5s, the number of base layers is 3, the exposure time of base layer is 7s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s; the exposure time of slot c is 6s, the number of base layers is 3, the exposure time of base layer is 8s, the peeling distance is 2mm, the peeling speed is 5mm / s, and the peeling recovery speed is 100mm / s. After printing, the model is peeled off from the deposition platform to obtain a multi-material DLP printed hydrogel scaffold product loaded with quercetin nanoparticles with light inhibition.

[0146] Figure 5 The printing resolution of the bio-ink using different flavonoid nanoparticles at 405nm (A) and 365nm (B) in Application Examples 1-8. The test was performed using 5% GelMA ink as a unified condition. As can be seen from the figure, when working at 405nm, except for the EGCG and GEN groups, the minimum resolution of the remaining groups can reach less than 100 microns; and when working at 365nm, the minimum resolution of the remaining groups can reach less than 80 microns. This reflects the excellent effect of using flavonoid nanoparticles as photoinhibitors. At the same time, due to the reaction mechanism of light-triggered free radical chain polymerization that is common to various light-based printings, flavonoid nanoparticle photoinhibitors can play a role in various light-based printings.

[0147] A Blank group was set up, and the addition of flavonoid nanoparticles was omitted based on Application Example 1.

[0148] Figure 6 The cell compatibility of the hydrogel scaffolds prepared in Example 1-8 and the Blank group on the 1st, 3rd and 5th day. It can be seen from the figure that various flavonoid nanoparticles have good cell compatibility. After endothelial cells were planted on the hydrogel scaffolds loaded with 0.1% flavonoid nanoparticles and cultured for 5 days, CCK8 detection showed that the flavonoid nanoparticles did not show cell inhibition at this concentration, and all groups were at the same level as the Blank group without flavonoid nanoparticles.

[0149] Figure 7The results of the antioxidant assay of the hydrogel scaffolds in Application Examples 1-8 and the Blank group, where A is a representative image after ROS probe labeling, and B is the normalized statistics of the green fluorescence intensity in the fluorescence photo, representing the intracellular ROS concentration in each group. After endothelial cells were planted on a hydrogel scaffold loaded with 0.1% flavonoid nanoparticles and cultured for 1 day, the cells were stimulated with hydrogen peroxide solution for 12 hours, and then labeled with ROS probe DCFH-DA and live cell nuclear dye Hoechst and photographed, and Merge is the superposition of DCFH-DA and Hoechst two-color fluorescence. As can be seen from the figure, the ROS of the Blank group without flavonoid nanoparticles is significantly stronger than that of other groups loaded with flavonoid nanoparticles, confirming that flavonoid nanoparticles can participate in the removal of intracellular ROS.

[0150] Figure 8 The actual printing performance of flavonoid nanoparticles in complex bioprinting scenarios. The figure shows the preparation effects of several scaffolds involved in the application examples, among which A is a product printed by high-precision SLA of a single material according to the method of application example 9, B is a product printed by high-precision DLP of cell-carrying ink according to the method of application example 10, C is a product printed by high-precision DLP of multiple materials according to the method of application example 11, and D is the result of constructing a bionic organ model according to the method of application example 11, which highlights the application value of flavonoid nanoparticles in biomedical engineering.

[0151] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A 3D printing light-blocking agent based on flavonoid nanoparticles, characterized in that: The raw materials include a flavonoid nanoparticle solution, wherein the flavonoid compound used in the preparation process of the flavonoid nanoparticle solution is one or more of rutin, epigallocatechin gallate, apigenin, genistein, hesperetin, quercetin, daidzein and naringenin; The method for preparing the flavonoid nanoparticle solution comprises the following steps: After dissolving the flavonoid compound in a NaOH solution, a buffer solution is added and mixed, and then concentrated hydrochloric acid is added to adjust the pH to obtain a hydrogel pre-liquid, and the hydrogel pre-liquid is filtered through a syringe filter to obtain the flavonoid nanoparticle solution; the buffer solution is a phosphate buffer solution.

2. The three-dimensional printing light-blocking agent based on flavonoid nanoparticles according to claim 1, characterized in that: The dosage ratio of the flavonoid compound to the buffer solution is (0.025-0.3) g: 3 mL.

3. The three-dimensional printing light-blocking agent based on flavonoid nanoparticles according to claim 1, characterized in that: Adjust pH to 7-9.

4. The three-dimensional printing light-blocking agent based on flavonoid nanoparticles according to claim 1, characterized in that: The raw materials also include a water-soluble photoinitiator, and the usage ratio of the flavonoid nanoparticle solution to the water-soluble photoinitiator is 10 mL: 0.025 g.

5. A method for preparing a 3D printing light-blocking agent based on flavonoid nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: The flavonoid nanoparticle solution is mixed and diluted with a buffer solution, and then a water-soluble photoinitiator is added, and after mixing, the three-dimensional printing light-blocking agent based on flavonoid nanoparticles is obtained.

6. The method for preparing a 3D printing light-blocking agent based on flavonoid nanoparticles according to claim 5, characterized in that: The flavonoid nanoparticle solution is mixed with a buffer solution and diluted to a concentration of 0.001-1.0 g / mL.

7. Use of the flavonoid nanoparticle-based three-dimensional printing light-blocking agent according to any one of claims 1 to 4 in the preparation of a photocurable scaffold material, characterized in that: The flavonoid nanoparticle-based three-dimensional printing light-blocking agent is mixed with a water-soluble photocurable resin to prepare a photosensitive ink solution, and the photosensitive ink solution is cured by light to obtain a photocurable bracket material.

8. The use of the flavonoid nanoparticle-based three-dimensional printing light-blocking agent in the preparation of a light-curing scaffold material according to claim 7, characterized in that: The photocuring shaping method includes ultraviolet light curing, photocuring in-situ 3D printing, SLA 3D printing, DLP 3D printing and two-photon lithography 3D printing.

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