Marine polysaccharide-based bio-ink for in-situ 3D printing as well as preparation method and application of marine polysaccharide-based bio-ink

Abstract

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

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] 3D bioprinting has attracted increasing attention due to its ability to print complex structures. Among them, 3D photocuring printing using UV polymerization technology has the advantages of high flexibility, high precision, and fast printing speed, and has been widely used in in vitro reconstruction studies of various tissues. However, surgical implantation has the risk of surgical complications and infection, which often causes secondary damage. If damaged tissue can be repaired in situ in a non-invasive way, iatrogenic damage will be greatly reduced. However, due to the poor tissue penetration depth, long-term exposure to ultraviolet light is harmful to biological tissues, and in situ repair of damaged tissues cannot be achieved using current photocuring printing technology. How to deliver light deeply into optically scattering media, such as biological tissues, remains technically challenging.

[0004] Compared with fluorescence imaging of ultraviolet light (<400nm), visible light (400-700nm) and the first near-infrared (NIR) window (NIR-I-700-900nm), the second NIR window (NIR-II, 900-1700nm) has stronger tissue penetration and is more suitable for deep biological tissues. The use of rare earth ion-doped luminescent composite materials can overcome the shortcomings of poor muscle penetration of ultraviolet light and damage to biological tissues. It can convert near-infrared II light into ultraviolet light of corresponding wavelengths in animals, realize in-situ 3D printing in vivo, and can be used as an ideal conversion material for light-activated in-situ 3D printing.

[0005] In addition, for 3D bioprinting, bio-ink, printing method and effective vascularization are the three main key technical challenges currently faced. It is currently difficult to design multifunctional printing materials with rheology, mechanics and bioactivity that meet clinical practice. Marine biomaterials are widely available, have low toxic side effects, high safety, biodegradability and molecular modifiability. Among them, polysaccharide-based marine biomaterials can not only simulate the extracellular matrix, but also have multiple biological activities such as antioxidant, anti-inflammatory, cell growth promotion, cell behavior regulation, and endothelial cell vascularization. However, there is still a lack of polysaccharide-based marine bio-inks suitable for in situ photocuring 3D printing. Therefore, the development of bio-inks with polysaccharide-based marine biomaterials as the main component that can be used for in situ photocuring 3D printing is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention 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 provided by the present invention has good mechanical properties and good biocompatibility, and can be used for in situ 3D photocuring bio-printing; and 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, vascular construction, tissue reconstruction, etc.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a marine polysaccharide-based biological ink, comprising the following preparation steps:

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

[0010] S2, mixed YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 O is heated to obtain the upconversion nanomaterial Na 3 ZF 7 :Yb,Tm;

[0011] S3, mixed with methacryloylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na dissolved in lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) 3 ZF 7 :Yb,Tm, and the marine polysaccharide-based bio-ink is obtained.

[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 the reaction is precipitated in pre-cooled ethanol, and then filtered and washed in sequence to obtain methacryloyl sodium alginate.

[0015] Preferably, in step S2, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 The molar ratio of O is 15-25:0.05-0.15:75-82.

[0016] Further preferably, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 The molar ratio of O is 20:0.1:79.9.

[0017] Preferably, in step S2, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 O is first dissolved in water, and then an organic solvent is added for heating reaction; further preferably, the organic solvent is NaOH and NH 4 F.

[0018] Preferably, in step S2, the heating reaction is carried out at a temperature of 150 to 250° C. and for a time of 10 to 15 hours.

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

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

[0021] Further preferably, the centrifugation time is 3 min; and the washing times are 3 times.

[0022] Preferably, in step S3, the methacrylylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na 3 ZF 7 :The mass volume concentration ratio of Yb and Tm is 4~6:3~7:2~5:3~5:10.

[0023] Preferably, the methacryloyl sodium alginate is the main component of the photocurable hydrogel, LAP is a photoinitiator, fucoidan has anti-inflammatory and antibacterial effects, collagen promotes angiogenesis, mussel mucin is conducive to cell adhesion and proliferation, and the upconversion nanomaterial Na 3 ZF 7 : The ultraviolet light emitted by Yb, Tm solidifies the hydrogel.

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

[0025] The third aspect of the present invention provides an application of the marine polysaccharide-based biological ink described in the second aspect in photocuring 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 the present invention uses natural marine biological materials as raw materials, which are abundant in source, low in cost, and have excellent biocompatibility.

[0028] (2) The upconversion material prepared by the present invention triggers the curing of bio-ink under 980nm excitation light, which can overcome the shortcomings of poor muscle penetration of ultraviolet light and damage to biological tissues. The stronger penetration of 980nm light source is conducive to the realization of in situ 3D printing in vivo, and is an ideal conversion material for light-activated in situ 3D printing.

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

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

[0031] (5) The marine polysaccharide-based bio-ink in the present invention has good molding properties, and the resulting hydrogel has high mechanical strength, which supports the printing of models with various shapes and structures. It has a wide range of applications and can be used for in situ tissue repair, vascular construction, tissue reconstruction, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the methacryloyl sodium alginate prepared in Example 1 of the present invention;

[0034] Figure 2 The up-conversion nanomaterial Na prepared in Example 1 of the present invention 3 ZF 7 : The emission spectrum of Yb, Tm and the absorption spectrum of the photoinitiator LAP, wherein (a) is the emission spectrum of the up-conversion luminescent nanomaterial, and (b) is the absorption spectrum of the photoinitiator LAP;

[0035] Figure 3 The up-conversion nanomaterial prepared in Comparative Example 1 of the present invention is NaYF 4 : The emission spectrum of Yb / Er and the absorption spectrum of the photoinitiator LAP, wherein (a) is the emission spectrum of the up-conversion luminescent nanomaterial, and (b) is the absorption spectrum of the photoinitiator LAP;

[0036] Figure 4 This is a photocured photograph of the marine polysaccharide-based biological ink prepared in Example 1 of the present invention;

[0037] Figure 5 The tissue penetration of the marine polysaccharide-based bio-ink prepared in Example 1 of the present invention;

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

[0039] Figure 7 The marine polysaccharide-based bio-ink prepared in Example 1 of the present invention promotes endothelial cell vascularization;

[0040] Figure 8 The 3D printing marine polysaccharide-based bio-ink prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] As mentioned above, there is still a lack of polysaccharide-based marine bio-inks suitable for in situ photocuring 3D printing. Therefore, developing bio-inks with polysaccharide-based marine biomaterials as the main components that can be used for in situ photocuring 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 a marine polysaccharide-based biological ink, comprising the following preparation steps:

[0044] S1, mixing sodium alginate and methacrylic anhydride to react to obtain methacrylated sodium alginate;

[0045] S2, mixed YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 O is heated to obtain the upconversion nanomaterial Na 3 ZF 7 :Yb,Tm;

[0046] S3, mixed with methacryloylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na dissolved in lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) 3 ZF 7 :Yb,Tm, and the marine polysaccharide-based bio-ink is obtained.

[0047] In one or more examples of this implementation mode, 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 examples of this embodiment, in step S1, the reaction is carried out under alkaline conditions, specifically, sodium hydroxide is added to the reaction solution to maintain the pH at 8.0.

[0049] In one or more examples of this embodiment, in step S1, the mixture after the reaction is precipitated in pre-cooled ethanol, and then filtered and washed in sequence to obtain methacryloyl sodium alginate.

[0050] In one or more examples of this embodiment, in step S2, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 The molar ratio of O is 15-25:0.05-0.15:75-82.

[0051] In one or more examples of this embodiment, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 The molar ratio of O is 20:0.1:79.9.

[0052] In one or more examples of this embodiment, in step S2, the YbCl 3 6H 2 O、TmCl 3 6H 2 O、Zr(NO 3 ) 4 ·5H 2 O is first dissolved in water, and then an organic solvent is added for heating reaction; further preferably, the organic solvent is NaOH and NH 4 F.

[0053] In one or more examples of this embodiment, 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 examples of this embodiment, in step S2, the heating reaction temperature is 200° C. and the time is 12 hours.

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

[0056] In one or more examples of this embodiment, the centrifugation time is 3 minutes; and the number of washing times is 3 times.

[0057] In one or more embodiments of this embodiment, in step S3, the methacrylylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na 3 ZF7 :The mass volume concentration ratio of Yb and Tm is 4~6:3~7:2~5:3~5:10.

[0058] In one or more embodiments of this embodiment, the methacryloyl sodium alginate is the main component of the photocurable hydrogel, LAP is a photoinitiator, fucoidan has anti-inflammatory and antibacterial effects, collagen promotes angiogenesis, mussel mucin is conducive to cell adhesion and proliferation, and the upconversion nanomaterial Na 3 ZF 7 : The ultraviolet light emitted by 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, which is 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 biological ink described in the second aspect in a photocuring printing technology.

[0061] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0062] Example 1 :

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

[0064] (1) Preparation of methacryloyl sodium alginate

[0065] Weigh 2g of sodium alginate into a round-bottom flask, add 100mL of purified water and stir at 50℃ for 1 hour. Then, drop 6mL of methacrylic anhydride at 0℃, and drop 0.5M NaOH solution to keep the pH of the mixture at 8.0, and stir for 12h. Precipitate the reactant in precooled ethanol, filter and purify by washing with precooled ethanol.

[0066] (2) Preparation of upconversion nanomaterials

[0067] Weigh 20mmol YbCl 3 6H 2 O, 0.1mmol TmCl 3 6H 2 O, 79.9mmolZr(NO 3 ) 4 ·5H 2 O in a beaker, add 10 mL of deionized water and stir evenly, and after it is completely dissolved, add 0.24 mol NaOH and 0.56 mol NH4 F was stirred for 30 min, and the reactants were transferred to a reactor and reacted in an oven at 200°C for 12 h. After the reaction, the mixture was centrifuged for 3 min and purified by washing with water and ethanol for 3 times to obtain the upconversion nanomaterial Na 3 ZF 7 :Yb,Tm.

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

[0069] Weigh an appropriate amount of LAP in a beaker and add PBS buffer solution, heat and dissolve in a water bath at 37°C for 30 minutes to obtain a 0.5 mg / mL LAP standard solution. Dissolve the prepared sodium methacryloyl alginate (5% w / v) in the LAP standard solution, slowly stir at 50°C, and add fucoidan (4% w / v), collagen (2.5% w / v) and mussel mucin (3% w / v), as well as the upconversion nanomaterial Na 3 ZF 7 :Yb,Tm(0.1g / mL), and obtained marine polysaccharide-based bio-ink that can respond to near-infrared light curing.

[0070] Example 2 :

[0071] This example characterizes the methacrylylated sodium alginate and upconversion luminescent nanomaterials prepared in Example 1:

[0072] like Figure 1 As shown, the H NMR spectrum showed that methacrylylated sodium alginate was successfully prepared;

[0073] like Figure 2 As shown, fluorescence tests show that the upconversion nanomaterial Na 3 ZF 7 :Yb,Tm emits ultraviolet light and can be absorbed by LAP.

[0074] Comparative Example 1 :

[0075] The difference from Example 1 is that the up-conversion nanomaterial used in this comparative example is NaYF 4 :Yb / Er, other preparation methods and parameters are the same as those in Example 1.

[0076] The results are as follows Figure 3 As shown, fluorescence tests show that the upconversion nanomaterial NaYF 4 :The emission light of Yb / Er cannot be absorbed by LAP, and thus the curing of bio-ink cannot be achieved.

[0077] Test Example 1 :

[0078] In this test example, the marine biopolysaccharide-based ink prepared in Example 1 was photocured.

[0079] Experimental process: Weigh an appropriate amount of upconversion nanomaterial Na 3 ZF 7 :Yb, Tm were dissolved in PBS buffer solution, mixed with marine polysaccharide-based bio-ink and stirred evenly, and then irradiated and cured with a 980nm laser to obtain a hydrogel.

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

[0081] Test Example 2 :

[0082] In this test example, the marine biopolysaccharide-based ink prepared in Example 1 was subjected to a tissue penetration test.

[0083] Experimental process: Cut chicken tissues with uniform thickness of 3mm, 6mm, and 10mm, add the marine polysaccharide-based bio-ink prepared in Example 1 into the well plate, place the chicken tissues on top of the well plate, and use a 980nm laser to penetrate the chicken tissues to solidify the hydrogel solution.

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

[0085] Test Example 3 :

[0086] In this test example, the marine polysaccharide-based bio-ink prepared in Example 1 was subjected to cell adhesion and proliferation experimental tests.

[0087] Experimental process: After the marine polysaccharide-based bio-ink is photocured, it is sterilized under ultraviolet light for 12 hours and transferred to a cell culture dish. The cell suspension is inoculated into the cell culture dish with the marine polysaccharide-based bio-ink. After the cells adhere to the wall, an appropriate volume of Calcein AM detection working solution is added and incubated at 37°C in the dark for 30 minutes. After the incubation, the staining effect is observed under a fluorescence microscope.

[0088] The results are as follows Figure 6 As shown, the bio-ink has the effect of promoting cell adhesion and proliferation.

[0089] Test Example 4 :

[0090] In this test example, the marine polysaccharide-based bio-ink prepared in Example 1 was used to perform an experimental test on promoting endothelial cell vascularization.

[0091] Experimental procedure: Inoculate the cell suspension into the cell culture dish with marine polysaccharide-based bio-ink and culture for 72 hours. Add an appropriate volume of Calcein AM detection working solution and incubate at 37°C in the dark for 30 minutes. After incubation, observe the staining effect under a fluorescence microscope.

[0092] The results are as follows Figure 7 As shown, the bio-ink promotes endothelial cells to connect into a lumen and promotes endothelial cell vascularization.

[0093] Application Example 1 :

[0094] In this application example, the marine polysaccharide-based bio-ink prepared in Example 1 is used for 3D printing.

[0095] Place the marine polysaccharide-based bio-ink in the material 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 is used for 3D printing to obtain models with different graphics.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing marine polysaccharide-based bio-ink for in-situ 3D printing, characterized in that: The method comprises the following preparation steps: S1, mixing sodium alginate and methacrylic anhydride to react to obtain methacrylated sodium alginate; S2, mixing YbCl3·6H2O, TmCl3·6H2O, and Zr(NO3)4·5H2O for heating reaction to obtain upconversion nanomaterial Na3ZrF7:Yb,Tm; S3, mixing methacryloylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na3ZrF7:Yb,Tm dissolved in phenyl (2,4,6-trimethylbenzoyl) lithium phosphate to obtain marine polysaccharide-based bio-ink.

2. The preparation method according to claim 1, characterized in that In step S1, the mass ratio of sodium alginate to methacrylic anhydride is 1:1 to 1:5, preferably 1:

3.

3. The preparation method according to claim 1, characterized in that: 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.

4. The preparation method according to claim 1, characterized in that: In step S1, the mixture after the reaction is precipitated in pre-cooled ethanol, and then filtered and washed in sequence to obtain methacryloyl sodium alginate.

5. The preparation method according to claim 1, characterized in that: 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; preferably, the molar ratio of YbCl3·6H2O, TmCl3·6H2O and Zr(NO3)4·5H2O is 20:0.1:79.

9.

6. The preparation method according to claim 1, characterized in that: 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 to carry out a heating reaction; preferably, the organic solvent is NaOH and NH4F.

7. The preparation method according to claim 6, characterized in that: The temperature of the heating reaction is 150-250°C, and the time of the heating reaction is 150-250°C for 10-15 hours; preferably, the temperature of the heating reaction is 200°C for 12 hours; After the heating reaction is completed, centrifugation and washing are performed in sequence; the centrifugation time is 2 to 5 minutes; the detergents used for washing are water and ethanol, and the number of washing times is 2 to 5 times; preferably, the centrifugation time is 3 minutes; the number of washing times is 3 times.

8. The preparation method according to claim 1, characterized in that: In step S3, the mass volume concentration ratio of the methacrylylated sodium alginate, fucoidan, collagen, mussel mucin and upconversion nanomaterial Na3ZrF7:Yb,Tm is 4-6:3-7:2-5:3-5:

10.

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

10. Use of the marine polysaccharide-based biological ink according to claim 9 in photocuring printing technology.

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