A marine polysaccharide-based bio-ink for in-situ 3D printing and a preparation method and application thereof

Abstract

The application belongs to the technical field of material preparation, and particularly relates to a marine polysaccharide-based bio-ink for in-situ 3D printing and a preparation method and application thereof. The marine polysaccharide-based bio-ink provided by the application forms a hydrogel with good mechanical properties and good biocompatibility, and can be used for in-situ 3D photocuring bio-printing. The hydrogel has the functions of promoting cell adhesion, proliferation, migration, anti-inflammation, anti-oxidation and promoting endothelial cell vascularization, and can be used for in-situ tissue repair, blood vessel construction, tissue reconstruction and the like, and has a high application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of material preparation, and particularly relates to a marine polysaccharide-based bio-ink for in-situ 3D printing and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the application without admitting that such information forms prior art.

[0003] 3D bioprinting has attracted more and more attention due to its ability to print complex structures. Among them, 3D light-cured printing using ultraviolet light polymerization technology has the advantages of high flexibility, high precision and fast printing speed, and has been widely used in the in-vitro reconstruction of various tissues. However, surgical implantation has the risk of surgical complications and infection, which often causes secondary damage. If the damaged tissue can be repaired in-situ by non-invasive means, it will greatly reduce iatrogenic damage. However, due to the difference in tissue penetration depth, long-term irradiation of ultraviolet light is harmful to biological tissues, and the current light-cured printing technology cannot achieve in-situ repair of damaged tissues. It is still challenging in technology to deliver light deep into optically scattering media such as biological tissues.

[0004] Compared with fluorescence imaging of ultraviolet light (<400 nm), visible light (400-700 nm) and the first near-infrared (NIR-I, 700-900 nm) window, the second NIR window (NIR-II, 900-1700 nm) has stronger tissue penetration and is more suitable for deep biological tissues. Using rare earth ion-doped luminescent composites, the disadvantages of poor muscle penetration of ultraviolet light and harm to biological tissues can be overcome, and the near-infrared light in the second region can be converted into ultraviolet light of corresponding wavelength in vivo, realizing in-situ 3D printing in vivo, which can be used as an ideal conversion material for photoactivated in-situ 3D printing.

[0005] In addition, for 3D bioprinting, bio-ink, printing method and effective vascularization are three major key technical challenges currently faced, and it is difficult to design multifunctional printing materials with rheology, mechanics and biological activity meeting clinical practice. Marine biomaterials are widely sourced, low in toxic and side effects, high in safety, and have biodegradability and molecular modifiability. Polysaccharide-based marine biomaterials not only can simulate extracellular matrix, but also have various biological activities such as antioxidant, anti-inflammatory, promoting cell growth, regulating cell behavior, and promoting endothelial cell vascularization. However, there is still a lack of polysaccharide-based marine bio-ink suitable for in-situ photopolymerization 3D printing, and therefore, it is an urgent technical problem to be solved in the field to develop polysaccharide-based marine bio-ink for in-situ photopolymerization 3D printing. SUMMARY

[0006] To solve the above technical problems, the purpose of the present application is to provide a marine polysaccharide-based bio-ink for in-situ 3D printing and a preparation method and application thereof. The hydrogel formed by the marine polysaccharide-based bio-ink has good mechanical properties and good biocompatibility, and can be used for in-situ 3D photopolymerization bioprinting. In addition, the hydrogel has the functions of promoting cell adhesion, proliferation, migration, anti-inflammatory, antioxidant and promoting endothelial cell vascularization, and can be used for in-situ tissue repair, vascular construction, tissue reconstruction and the like.

[0007] To achieve the above purpose, the present application is realized by the following technical scheme:

[0008] In a first aspect, the present application provides a preparation method of marine polysaccharide-based bio-ink, comprising the following preparation steps:

[0009] S1, mixing sodium alginate and methacrylic anhydride to react to obtain methacrylated sodium alginate;

[0010] S2, mixing YbCl3·6H2O, TmCl3·6H2O and Zr(NO3)4·5H2O to react under heating to obtain upconversion nanomaterial Na3ZrF7:Yb,Tm;

[0011] S3, mixing methacrylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na3ZrF7:Yb,Tm dissolved in lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP) to obtain marine polysaccharide-based bio-ink.

[0012] Preferably, in step S1, the mass ratio of sodium alginate to methacrylic anhydride is 1:1 to 1:5, preferably 1:3.

[0013] Preferably, in step S1, the reaction is carried out under alkaline conditions, specifically by adding sodium hydroxide to the reaction solution to maintain the pH at 8.0.

[0014] Preferably, in step S1, the mixture after reaction is precipitated in pre-cooled ethanol, and then filtered and washed sequentially to obtain sodium methacrylamide.

[0015] Preferably, in step S2, the molar ratio of YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O is 15-25:0.05-0.15:75-82.

[0016] More preferably, the molar ratio of YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O is 20:0.1:79.9.

[0017] Preferably, in step S2, the YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O are first dissolved in water, and then an organic solvent is added for heating reaction; more preferably, the organic solvent is NaOH and NH4F.

[0018] Preferably, in step S2, the heating reaction temperature is 150–250°C and the time is 10–15 h.

[0019] More preferably, in step S2, the heating reaction temperature is 200°C and the time is 12 hours.

[0020] Preferably, in step S2, after the heating reaction is completed, centrifugation and washing are performed sequentially; the centrifugation time is 2 to 5 minutes; the washing agent used is water and ethanol, and the washing is performed 2 to 5 times.

[0021] More preferably, the centrifugation time is 3 minutes; and the washing is performed 3 times.

[0022] Preferably, in step S3, the mass-to-volume concentration ratio of the sodium methacrylamide, fucoidan, collagen, mussel adhesive protein, and upconversion nanomaterial Na3ZrF7:Yb,Tm is 4-6:3-7:2-5:3-5:10.

[0023] Preferably, the sodium methacrylamide alginate is the main component of the photocurable hydrogel, LAP is the photoinitiator, fucoidan has anti-inflammatory and antibacterial effects, collagen promotes angiogenesis, mussel adhesive protein is beneficial to cell adhesion and proliferation, and the ultraviolet light emitted by the upconversion nanomaterial Na3ZrF7:Yb,Tm solidifies the hydrogel.

[0024] In a second aspect, the present invention provides a marine polysaccharide-based bio-ink for in-situ 3D printing, obtained by the preparation method described in the first aspect.

[0025] In a third aspect, the present invention provides an application of the marine polysaccharide-based bio-ink described in the second aspect in photopolymerization printing technology.

[0026] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0027] (1) The marine polysaccharide-based bio-ink prepared by this invention is made from natural marine biological materials, which are abundant, low in cost, and have excellent biocompatibility.

[0028] (2) The upconversion material prepared by the present invention can trigger the curing of bio-ink under 980nm excitation light, which can overcome the disadvantages of poor penetration of ultraviolet light into muscles and damage to biological tissues. The 980nm light source has stronger penetration, which is conducive to in-situ 3D printing in biological tissues. It is an ideal conversion material for photoactivated in-situ 3D printing.

[0029] (3) This invention provides a marine polysaccharide-based bio-ink that can simulate the environment of the extracellular matrix, providing a good environment for the in vitro three-dimensional culture of cells, and can respond to near-infrared light curing to achieve in-situ printing that penetrates 10mm of biological tissue, overcoming the problem that current photocuring printing technology cannot achieve in-situ curing that penetrates tissue.

[0030] (4) The marine polysaccharide-based bio-ink in this invention has the ability to promote cell growth, tissue repair, anti-inflammation and angiogenesis, which can solve the problems of biosafety and bioactivity of most hydrogels.

[0031] (5) The marine polysaccharide-based bio-ink of the present invention has good molding performance and produces hydrogel with high mechanical strength. It supports the printing of models with various shapes and structures and has a wide range of applications, including in situ tissue repair, blood vessel construction, and tissue reconstruction. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The 1H NMR spectrum of sodium methacrylamide prepared in Example 1 of this invention;

[0034] Figure 2The emission spectrum of the upconversion nanomaterial Na3ZrF7:Yb,Tm prepared in Example 1 of this invention and the absorption spectrum of the photoinitiator LAP are shown, where (a) is the emission spectrum of the upconversion luminescent nanomaterial and (b) is the absorption spectrum of the photoinitiator LAP.

[0035] Figure 3 The emission spectrum of the upconversion nanomaterial NaYF4:Yb / Er prepared in Comparative Example 1 of this invention and the absorption spectrum of the photoinitiator LAP are shown, where (a) is the emission spectrum of the upconversion luminescent nanomaterial and (b) is the absorption spectrum of the photoinitiator LAP.

[0036] Figure 4 Photocured image of marine polysaccharide-based bio-ink prepared in Example 1 of this invention;

[0037] Figure 5 Tissue penetration of the marine polysaccharide-based bioink prepared in Example 1 of this invention;

[0038] Figure 6 Cell adhesion and proliferation of the marine polysaccharide-based bioink prepared in Example 1 of this invention;

[0039] Figure 7 Promoting endothelial cell angiogenesis of the marine polysaccharide-based bioink prepared in Example 1 of this invention;

[0040] Figure 8 This is the 3D printing marine polysaccharide-based bio-ink prepared in Example 1 of the present invention. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] As mentioned above, there is still a lack of polysaccharide-based marine bio-inks suitable for in-situ photopolymerization 3D printing. Therefore, developing bio-inks with polysaccharide-based marine biomaterials as the main component that can be used for in-situ photopolymerization 3D printing is a technical problem that urgently needs to be solved in this field.

[0043] A first typical embodiment of the present invention provides a method for preparing marine polysaccharide-based bio-ink, comprising the following preparation steps:

[0044] S1. Sodium alginate and methacrylic anhydride are reacted to obtain methacrylated sodium alginate;

[0045] S2, YbCl3·6H2O, TmCl3·6H2O and Zr(NO3)4·5H2O were mixed and heated to obtain the upconversion nanomaterial Na3ZrF7:Yb,Tm;

[0046] S3. A mixture of sodium methacryloylated alginate, fucoidan, collagen, mussel adhesive protein, and upconversion nanomaterial Na3ZrF7:Yb,Tm dissolved in phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) is used to obtain marine polysaccharide-based bio-ink.

[0047] In one or more embodiments of this implementation, in step S1, the mass ratio of sodium alginate to methacrylic anhydride is 1:1 to 1:5, preferably 1:3.

[0048] In one or more embodiments of this implementation, in step S1, the reaction is carried out under alkaline conditions, specifically by adding sodium hydroxide to the reaction solution to maintain the pH at 8.0.

[0049] In one or more embodiments of this implementation, in step S1, the reacted mixture is precipitated in pre-cooled ethanol, and then sequentially filtered and washed to obtain sodium methacrylamide.

[0050] In one or more embodiments of this implementation, in step S2, the molar ratio of YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O is 15-25:0.05-0.15:75-82.

[0051] In one or more embodiments of this implementation, the molar ratio of YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O is 20:0.1:79.9.

[0052] In one or more embodiments of this implementation, in step S2, the YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O are first dissolved in water, and then an organic solvent is added for heating reaction; more preferably, the organic solvent is NaOH and NH4F.

[0053] In one or more embodiments of this implementation, in step S2, the temperature of the heating reaction is 150-250°C and the time is 10-15 hours.

[0054] In one or more embodiments of this implementation, in step S2, the heating reaction temperature is 200°C and the time is 12 hours.

[0055] In one or more embodiments of this implementation, in step S2, after the heating reaction is completed, centrifugation and washing are performed sequentially; the centrifugation time is 2 to 5 minutes; the washing agent used is water and ethanol, and the washing is performed 2 to 5 times.

[0056] In one or more embodiments of this implementation, the centrifugation time is 3 minutes; the washing is performed 3 times.

[0057] In one or more embodiments of this implementation, in step S3, the mass-volume concentration ratio of the sodium methacrylamide, fucoidan, collagen, mussel adhesive protein, and upconversion nanomaterial Na3ZrF7:Yb,Tm is 4-6:3-7:2-5:3-5:10.

[0058] In one or more embodiments of this implementation, sodium methacrylamide is the main component of the photocurable hydrogel, LAP is a photoinitiator, fucoidan has anti-inflammatory and antibacterial effects, collagen promotes angiogenesis, mussel adhesive protein is beneficial for cell adhesion and proliferation, and the ultraviolet light emitted by the upconversion nanomaterial Na3ZrF7:Yb,Tm solidifies the hydrogel.

[0059] A second typical embodiment of the present invention provides a marine polysaccharide-based bio-ink for in-situ 3D printing, obtained by the preparation method described in the first aspect.

[0060] A third typical embodiment of the present invention provides an application of the marine polysaccharide-based bio-ink described in the second aspect in photopolymerization printing technology.

[0061] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0062] Example 1 :

[0063] This embodiment provides a method for preparing marine polysaccharide-based bio-ink for in-situ 3D printing, including the following preparation steps:

[0064] (1) Preparation of sodium methacrylamide

[0065] Weigh 2g of sodium alginate into a round-bottom flask, add 100mL of purified water, and stir at 50℃ for 1 hour. Then, add 6mL of methacrylic anhydride dropwise at 0℃, while simultaneously adding 0.5M NaOH solution to maintain the pH of the mixture at 8.0, and stir for 12 hours. Precipitate the reaction mixture in pre-cooled ethanol, filter, and wash with pre-cooled ethanol to purify.

[0066] (2) Preparation of upconversion nanomaterials

[0067] Weigh 20 mmol YbCl3·6H2O, 0.1 mmol TmCl3·6H2O, and 79.9 mmol Zr(NO3)4·5H2O into a beaker, add 10 mL of deionized water and stir until completely dissolved. Then add 0.24 mol NaOH and 0.56 mol NH4F and stir for 30 min. Transfer the reactants to a reaction vessel and react at 200 °C for 12 h in an oven. After the reaction is complete, centrifuge for 3 min and wash three times with water and ethanol respectively to obtain the upconversion nanomaterial Na3ZrF7:Yb,Tm.

[0068] (3) Preparation of marine polysaccharide-based bio-ink

[0069] Weigh an appropriate amount of LAP into a beaker, add PBS buffer solution, and heat in a water bath at 37°C for 30 min to dissolve, obtaining a 0.5 mg / mL LAP standard solution. Dissolve the prepared sodium methacrylamide (5% w / v) in the LAP standard solution, stir slowly at 50°C until completely dissolved, then add fucoidan (4% w / v), collagen (2.5% w / v), mussel adhesive protein (3% w / v), and upconversion nanomaterial Na3ZrF7:Yb,Tm (0.1 g / mL) to obtain a marine polysaccharide-based bio-ink that can be cured in response to near-infrared light.

[0070] Example 2 :

[0071] This embodiment characterizes the sodium methacrylamide and upconversion luminescent nanomaterials prepared in Example 1:

[0072] like Figure 1 As shown, the 1H NMR spectrum indicates that sodium methacrylamide was successfully prepared;

[0073] like Figure 2 As shown, fluorescence testing indicates that the upconversion nanomaterial Na3ZrF7:Yb,Tm emits ultraviolet light and can be absorbed by LAP.

[0074] Comparative Example 1 :

[0075] The difference from Example 1 is that the upconversion nanomaterial used in this comparative example is NaYF4:Yb / Er, and the other preparation methods and parameters are the same as in Example 1.

[0076] The results are as follows Figure 3 As shown, fluorescence testing indicates that the emitted light from the upconversion nanomaterial NaYF4:Yb / Er cannot be absorbed by the LAP, thus preventing the curing of the bio-ink.

[0077] Test Example 1 :

[0078] This experimental example demonstrates the photocuring of the marine biological polysaccharide-based ink prepared in Example 1.

[0079] Experimental procedure: Weigh an appropriate amount of upconversion nanomaterial Na3ZrF7:Yb,Tm and dissolve it in PBS buffer solution. Mix it with marine polysaccharide-based bio-ink and stir evenly. After curing by irradiation with a 980nm laser, a hydrogel is obtained.

[0080] The results are as follows Figure 4 As shown, the hydrogel successfully solidified under 980nm light irradiation, and no flowing components were found when the vial was inverted.

[0081] Test Example 2 :

[0082] This experimental example demonstrates the tissue penetration test of the marine biological polysaccharide-based ink prepared in Example 1.

[0083] Experimental procedure: Chicken tissue of uniform thickness of 3 mm, 6 mm and 10 mm was cut, and marine polysaccharide-based bio-ink prepared in Example 1 was added into the well plate. The chicken tissue was placed on the well plate, and a 980 nm laser penetrated the chicken tissue to solidify the hydrogel solution.

[0084] The results are as follows Figure 5 As shown, 980nm light can penetrate up to 10mm thick chicken tissue to solidify the bio-ink.

[0085] Test Example 3 :

[0086] This experimental example tests the cell adhesion and proliferation of the marine polysaccharide-based bioink prepared in Example 1.

[0087] Experimental Procedure: After photocuring, the marine polysaccharide-based bio-ink was sterilized under UV light for 12 hours and then transferred to cell culture dishes. Cell suspension was inoculated into the cell culture dishes containing the marine polysaccharide-based bio-ink. After cell adhesion, an appropriate volume of Calcein AM detection working solution was added, and the mixture was incubated at 37°C in the dark for 30 minutes. After incubation, the staining effect was observed under a fluorescence microscope.

[0088] The results are as follows Figure 6 As shown, this bio-ink promotes cell adhesion and proliferation.

[0089] Test Example 4 :

[0090] This experimental example demonstrates the endothelial cell angiogenesis promotion experiment of the marine polysaccharide-based bioink prepared in Example 1.

[0091] Experimental procedure: Cell suspension was seeded into cell culture dishes containing marine polysaccharide-based bio-ink and cultured for 72 h. An appropriate volume of Calcein AM detection working solution was added, and the mixture was incubated at 37°C in the dark for 30 min. After incubation, the staining effect was observed under a fluorescence microscope.

[0092] The results are as follows Figure 7 As shown, this bio-ink promotes the connection of endothelial cells into tubular structures and promotes endothelial cell vascularization.

[0093] Application Example 1 :

[0094] This application example demonstrates the use of the marine polysaccharide-based bio-ink prepared in Example 1 for 3D printing.

[0095] Place the marine polysaccharide-based bio-ink into the feed tank of the 3D printer, select the printing model, set the speed to 8 mm / s and the air pressure to 0.12 MPa for 3D printing.

[0096] like Figure 8 As shown, the marine polysaccharide-based bio-ink prepared in Example 1 can be used for 3D printing to obtain models with different shapes.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the preparation of marine polysaccharide-based bio-ink for in situ 3D printing, characterized in that, The preparation method comprises the following steps: S1, mixing sodium alginate and methacrylic anhydride to react, dropping methacrylic anhydride at 0℃ to obtain methacrylated sodium alginate; S2, YbCl3 6H2O, TmCl3 6H2O, Zr(NO3)4 5H2O, to obtain the upconversion nanomaterial Na3ZrF7:Yb,Tm; S3, mixing methacrylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na3ZrF7:Yb,Tm dissolved in lithium phenyl(2,4,6-trimethylbenzoyl)phosphate to obtain the marine polysaccharide-based bio-ink; In step S2, the YbCl3 6H2O, TmCl3 6H2O, Zr(NO3)4 5H2O is first dissolved in water, and then an organic solvent is added for heating reaction.

2. The production method according to claim 1, wherein In step S1, the mass ratio of sodium alginate to methacrylic anhydride is 1:1-1:

5.

3. The production method according to claim 2, wherein In step S1, the mass ratio of sodium alginate to methacrylic anhydride is 1:

3.

4. The production method according to claim 1, wherein In step S1, the reaction is carried out under alkaline conditions, specifically, sodium hydroxide is added to the reaction solution to keep pH at 8.

0.

5. The production method according to claim 1, wherein In step S1, the mixture after reaction is precipitated in pre-cooled ethanol, and then is subjected to suction filtration and washing in sequence to obtain methacrylated sodium alginate.

6. The production method according to claim 1, wherein In step S2, the YbCl3 6H2O, TmCl3 6H2O, Zr(NO3)4 The molar ratio of 5H2O is 15-25:0.05-0.15:75-82.

7. The production method according to claim 6, wherein In step S2, the YbCl3 6H2O, TmCl3 6H2O, Zr(NO3)4 The molar ratio of YbCl3:6H2O:5H2O is 20:0.1:79.

9.

8. The production method according to claim 1, wherein In step S2, the organic solvent is NaOH and NH4F.

9. The production method according to claim 1, wherein The heating reaction is carried out at a temperature of 150-250℃ for 10-15 h; after the heating reaction, centrifugation and washing are carried out in sequence; the centrifugation is carried out for 2-5 min; the washing is carried out with water and ethanol, and the washing is carried out for 2-5 times.

10. The production method according to claim 9, wherein The heating reaction is carried out at a temperature of 200℃ for 12 h; the centrifugation is carried out for 3 min; the washing is carried out for 3 times.

11. The production method according to claim 1, wherein In step S3, the mass-volume concentration ratio of methacrylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na3ZrF7:Yb,Tm is 4-6:3-7:2-5:3-5:

10.

12. A marine polysaccharide-based bio-ink for in situ 3D printing, characterized in that, The marine polysaccharide-based bio-ink is obtained by the preparation method of any one of claims 1-8.

13. Use of the marine polysaccharide-based bio-ink of claim 12 in light-cured printing technology.

Images