Femtosecond laser two-photon induced photocuring hydrogel and preparation method thereof

Abstract

The invention belongs to the field of special materials for biomedical additive manufacturing, and discloses femtosecond laser two-photon induced photocuring hydrogel and a preparation method thereof, and the hydrogel comprises the following components: high-substitution-degree methylacryloyl gelatin (GelMA), a photoinitiator P2CK, a polymer PEGDA and a PBS buffer solution. The method is technically characterized in that in the hydrogel, methylacryloyl gelatin (GelMA) is used as a hydrogel precursor, P2CK is used as a special two-photon initiator, a polymer PEGDA is used as a biological cross-linking agent, a PBS buffer solution is used as a gel precursor solvent, and a high-mechanical-strength and high-resolution hydrogel structure is prepared by using a femtosecond laser two-photon polymerization technology. The chemical synthesis method is characterized in that methacrylation of the transparent glue is carried out in a sodium carbonate buffer solution, and P2CK is formed by condensation of cyclopentanone and benzaldehyde 3-[(4-formylphenyl)-methylamino] propionate aldol. The hydrogel disclosed by the invention is high in two-photon polymerization efficiency, high in processing resolution and strong in mechanical property, and has a wide application prospect in the biomedical field.

Description

Technical Field

[0001] The present invention belongs to the field of special materials for biomedical additive manufacturing, and particularly relates to a femtosecond laser two-photon induced photocurable hydrogel and a preparation method thereof. Background Art

[0002] Femtosecond laser two-photon polymerization (TPP) can utilize the optical nonlinear absorption effect to induce the polymerization or cross-linking of photosensitive materials, realize the arbitrary construction of true three-dimensional micro-nano structures, and achieve a manufacturing resolution beyond the optical diffraction limit. It is an important processing method for microcellular scaffolds, bioprostheses, in-vivo robots, and drug capsule shells, and has important applications in the field of biomedicine.

[0003] Existing photocurable hydrogels can be divided into two categories: synthetic hydrogels and natural-derived hydrogels. Among them, synthetic hydrogels are mainly formed by using synthetic materials such as PEG, PVA, and poly(2-hydroxyethyl methacrylate) (PHEMA) as precursors and adopting a photocuring method. Synthetic hydrogel materials have high biotoxicity and strong irritation to the human body, and have not been widely approved for use in the human body so far.

[0004] Most natural-derived hydrogels are derived from natural extracellular matrices and have good biocompatibility, which can provide better biochemical and biological cues for surrounding cells. In particular, methacryloyl gelatin (GelMA) inherits the biological activity of gelatin, which is the main component of the natural extracellular matrix, and has become the gold standard in the field of biomaterials. However, due to post-treatment distortion caused by poor mechanical properties, even the hydrogel structure prepared using a relatively high concentration of GelMA (20%) cannot exceed the sub-micron resolution. Some strategies have been used to improve the mechanical properties of GelMA hydrogels. For example, manufacturing a hydrogel structure on a scaffold made of a stronger material, or further methacryloylation of the carboxylic acids present in GelMA to increase the number of photopolymerizable functional groups. However, the former introduces cytotoxic materials and destroys the biocompatibility of the hydrogel, while the latter requires complex chemical reactions and is difficult to synthesize materials. In addition, although hydrogel structures made from a photosensitive solution composed of GelMA and PEGDA with a DS of about 70% have been reported. However, due to the insufficient degree of substitution of GelMA, the strength of the hydrogel structure is too low and the deformation is serious. Other natural-derived materials, such as hyaluronic acid, chitosan, sodium alginate, etc., the hydrogel structures formed by them alone or in combination with GelMA do not have significantly better strength than the hydrogels formed only by GelMA. Thus, the low structural resolution and serious deformation of natural-derived hydrogels due to insufficient strength have hindered their application in the field of bioadditive manufacturing.

[0005] Therefore, there is an urgent need to develop a femtosecond laser two-photon induced photocurable hydrogel and its preparation method, so that it has the characteristics of high strength, high precision, and excellent biocompatibility, in order to promote the development of the field of special materials for biomedical additive manufacturing. Summary of the Invention

[0006] To solve the above existing problems, the present invention proposes a hydrogel with DS≥100 GelMA as the main body.

[0007] The synthesis method of GelMA in the hydrogel is as follows: At 50 °C, gelatin type B with a bloom value of 250 is dissolved in 25M CB buffer at a concentration of 10 w / v%, and the pH is adjusted to the range of 8.5 - 9.0 with sodium hydroxide or hydrochloric acid. Then, 94% methacrylic anhydride (MAA) is added to the above solution. The reaction lasts for 4 hours, and then the pH is adjusted to 7.2 - 7.4 to stop the reaction. The whole process is carried out under magnetic stirring at 500 rpm. After filtration, dialysis and freeze-drying, the product is stored at -20 °C for further experiments. The DS of GelMA is obtained by 1H-NMR measurement.

[0008] The synthesis method of the photoinitiator P2CK in the hydrogel is as follows: Before precipitation in ethanol / hydrogen chloride solution, freshly distilled cyclopentanone and benzaldehyde 3-[(4-formylphenyl)-methylamino]propionic acid are refluxed in NaOH solution for 6 hours for sufficient aldol condensation. After washing with cold methanol, the product is dried in vacuo to obtain the final product.

[0009] A femtosecond laser two-photon induced photocurable hydrogel and its preparation method are explored. The raw materials include: GelMA hydrogel precursor with DS≥100, photoinitiator P2CK, crosslinking agent polyethylene glycol diacrylate (PEGDA, Mn = 400 - 1200), and solvent 1×PBS buffer. It is characterized in that: The proportion of GelMA in the solution is 30 - 45% (w / v); the proportion of P2CK in the solution is 2 - 5 mol% (relative to the amount of double bonds present); the proportion of PEGDA (Mn = 400 - 1000) in the solution is 2 - 8% (v / v); the 1×PBS buffer is used as the solvent for the hydrogel precursor.

[0010] A femtosecond laser two-photon induced photocurable hydrogel is prepared, and it is characterized in that: (1) Add P2CK to a centrifuge tube, and then use a pipette to add PBS buffer to this centrifuge tube and shake for 10 - 15 minutes; (2) Add GelMA into the same centrifuge tube, dissolve it in PBS buffer solution, and heat the centrifuge tube in a constant temperature water bath at 37 - 50 °C for 1 - 2 hours; during this period, use an ultrasonic cleaner to ultrasonically oscillate the centrifuge tube 3 times, 10 minutes each time; (3) After adding PEGDA, heat the centrifuge tube in a water bath for 30 - 50 minutes, and oscillate it 3 times during this period; (4) Filter the above solution with a 0.22 μm needle - type filter.

[0011] The femtosecond laser two - photon polymerization processing characteristics of the prepared hydrogel were studied, and it is characterized in that: Use femtosecond laser for two - photon polymerization micro - nano processing in the prepared hydrogel, and use the strategies of changing the scanning speed with a fixed laser power and changing the laser power with a fixed scanning speed to study the structural ultimate processing resolution.

[0012] The present invention prepared a femtosecond laser two - photon induced photocurable hydrogel and used it to manufacture 3D structures. The structure shows higher strength than any structure manufactured by previous GelMA - based hydrogels. Rheological tests show that the hydrogel of the present invention is more than 10 times higher than traditional hydrogels of the same type in terms of storage modulus, reaching 4.7×10 5 Pa. The present invention combines femtosecond laser direct writing processing as a high - resolution additive manufacturing technology with GelMA as a natural derivative material with the most promising biomedical applications, providing a new method. Description of the Drawings

[0013] Figure 1 Schematic diagram of the synthesis of GelMA (methacryloyl gelatin);

[0014] Figure 2 Top view of the SEM image of the polymer line manufactured by single - scan with a constant scanning speed of 30 μm / s and different powers;

[0015] Figure 3 SEM image of the cross - beam (laser power 23 mW, scanning speed 19 mm / s) obtained in the resolution limit exploration;

[0016] Figure 4 Comparison of the storage modulus between the hydrogel of the present invention and the DS90 - GelMA room - temperature limit solubility hydrogel (results of photo - rheological tests), showing that the hydrogel of the present invention is more than 10 times higher than traditional hydrogels of the same type in terms of storage modulus. Detailed Embodiments

[0017] The following further describes the present invention in detail in combination with specific embodiments. Synthesis of high - substitution - degree GelMA: At 50 °C, type B gelatin with a bloom value of 250 was dissolved in 25 M CB buffer at a concentration of 10 w / v%, and the pH was adjusted to 9.0 with sodium hydroxide or hydrochloric acid. Then, 94% methacrylic anhydride (MAA) was added to the above solution. The reaction continued for 4 hours, and then the pH was adjusted to 7.4 to stop the reaction. The whole process was carried out under magnetic stirring at 500 rpm. After filtration, dialysis, and freeze-drying, the product was stored at -20 °C for further experiments. The DS of GelMA was obtained by 1H-NMR measurement.

[0018] Synthesis of initiator P2CK: Before precipitation in ethanol / hydrogen chloride solution, freshly distilled cyclopentanone and 3-[(4-formylphenyl)-methylamino] propionic acid benzaldehyde were refluxed in NaOH solution for 6 hours for sufficient aldol condensation. After washing with cold methanol, the product was dried in vacuo to obtain the final product.

[0019] Preparation process of hydrogel: (1) Add P2CK to a centrifuge tube, and then use a pipette to add PBS buffer to the centrifuge tube and shake for 10 minutes; (2) Add GelMA to the same centrifuge tube, dissolve it in PBS buffer, and heat the centrifuge tube in a constant temperature water bath at 37 °C for 60 minutes; during this period, use an ultrasonic cleaner to ultrasonically oscillate the centrifuge tube 3 times, 10 minutes each time; (3) After adding PEGDA, heat the centrifuge tube in a water bath for 30 minutes and oscillate 3 times during this period; (4) Filter the above solution with a 0.22 μm needle filter.

[0020] Processing process of hydrogel: (1) Place the hydrogel in the cavity of a silicone elastomeric ring clamped between two cover glasses to prevent solution evaporation. The bottom glass plate was silanized with 3-(trimethoxysilyl)propyl methacrylate to improve the bonding strength between the polymer structure and the glass plate; (2) Fix the glass slide on a two-photon three-dimensional moving processing platform, and focus the femtosecond laser on the hydrogel by means of downward exposure. Selective curing is achieved through the relative displacement path set by the laser focus in the hydrogel; (3) First, set a fixed scanning speed of 6 mm / s, and the power varies from 30 mW to 1 mW to explore the resolution limit of the cross-beam structure. Then, set a fixed power of 23 mW, and the scanning speed varies from 1 mm / s to 25 mm / s to explore the resolution limit of the cross-beam structure; (4) Manufacture polymer lines by single scanning at a constant scanning speed of 30 μm / s and different powers; After processing using femtosecond laser direct writing technology, the structure was placed in a PBS buffer solution at 37 °C for more than 30 hours and soaked in deionized water for 10 minutes. The structure was obtained by freeze-drying in a vacuum freeze dryer for 24 h.

[0021] The preferred embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0022] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0023] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A femtosecond laser two-photon induced photocurable hydrogel and its preparation method, Characterized in that: The raw materials include: GelMA hydrogel precursor with a degree of substitution (DS) ≥ 100, photoinitiator P2CK, polyethylene glycol diacrylate (PEGDA), 1×PBS buffer. The GelMA is used as the hydrogel precursor in the hydrogel, and the proportion in the hydrogel is 30-45% (w / v); the P2CK is used as the initiator in the hydrogel, and the proportion in the hydrogel is 2-5 mol% (relative to the amount of double bonds present); the PEGDA (Mn = 400-1200) is used as the crosslinking agent in the hydrogel, and the proportion in the hydrogel is 2-8% (v / v); the 1×PBS buffer is used as the solvent of the hydrogel.

2. The synthesis method of DS≥100 GelMA in claim 1, Characterized in that: At 50 °C, type B gelatin with a bloom value of 250 is dissolved in 25M CB buffer at a concentration of 10 w / v%, and the pH is adjusted to the range of 8.5-9.0 with sodium hydroxide or hydrochloric acid. Then, 94% of methacrylic anhydride (MAA) is added to the above solution. The reaction lasts for 4 hours, and then the pH is adjusted to 7.2-7.4 to stop the reaction. The whole process is carried out under magnetic stirring at 500 rpm. After filtration, dialysis and freeze-drying, the product is stored at -20 °C for further experiments. The DS of GelMA is obtained by 1H-NMR measurement.

3. The synthesis method of the photoinitiator P2CK in claim 1, Characterized in that: Before precipitation in ethanol / hydrogen chloride solution, freshly distilled cyclopentanone and benzaldehyde 3-[(4-formylphenyl)-methylamino]propionic acid are refluxed in NaOH solution for 6-8 hours for sufficient aldol condensation. After washing with cold methanol, the product is dried in vacuo to obtain the final product.

4. The preparation method of the hydrogel in claim 1, Characterized in that: (1) Add P2CK to a centrifuge tube, and then use a pipette to add PBS buffer to the centrifuge tube and shake for 10-15 minutes; (2) Add GelMA to the same centrifuge tube, dissolve it in PBS buffer, and heat the centrifuge tube in a constant temperature water bath at 37-50 °C for 1-2 hours; during this period, use an ultrasonic cleaner to ultrasonically oscillate the centrifuge tube 3 times, 10 minutes each time; (3) After adding PEGDA, heat the centrifuge tube in a water bath for 30-50 minutes and oscillate 3 times during this period; (4) Filter the above solution with a 0.22 μm needle filter.

5. A femtosecond laser two-photon induced photocurable hydrogel and its preparation method in claim 1, Characterized in that: The femtosecond laser parameters are: wavelength is 700-1000 nm; laser power ≥ 5 mW; laser scanning speed ≤ 20000 μm / s, and the final structure is obtained by freeze-drying.

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