A method for preparing an acoustic assembly multilayer structure based on a hydrophobic cavity

Abstract

This invention belongs to the field of biomanufacturing technology, specifically relating to a method for preparing an acoustically assembled multilayer structure based on a hydrophobic chamber. The method includes the following steps: adding a photocurable aqueous assembly liquid containing dispersed assembly units to a hydrophobic chamber, with the liquid level below the outer edge of the chamber; the sidewalls of the hydrophobic chamber are made of a hydrophobic material; under Faraday wave assembly drive, the assembly units form a specific patterned structure and are then fixed to obtain a cured surface layer; the above steps are repeated until a multilayer structure with the same thickness as the hydrophobic chamber is obtained. This invention addresses the influence mechanism of the assembly process and proposes a scheme to replace the polymethyl methacrylate chamber with a hydrophobic chamber, overcoming the limitation that the assembly liquid must fill the chamber completely.

Description

Technical Field

[0001] This invention belongs to the field of biomanufacturing technology, specifically relating to a method for preparing an acoustically assembled multilayer structure based on a hydrophobic cavity. Background Technology

[0002] Acoustic bioassembly utilizes sound waves to assemble pre-formed modular units, including cells or cell aggregates, and cell-biomaterial composites, to create biomimetic multi-layered structures. As an emerging bioassembly technology, acoustic bioassembly possesses numerous advantages and enormous application potential. Faraday waves, a type of sound wave, are defined as nonlinear standing waves caused by acceleration generated by the vertical oscillation of a liquid layer, first proposed by the British physicist Michael Faraday in 1831. Chen et al. first proposed applying Faraday waves to bioassembly, using them to assemble polystyrene microspheres and cell-carrying microspheres. By adjusting assembly parameters such as vibration frequency, amplitude, and the shape and size of the assembly chamber, different patterned structures were obtained within seconds. The Faraday wave bioassembly method, through the acoustic radiation force or fluid force generated by sound waves, can assemble modules within a field within seconds. It features non-contact, non-invasive, and highly efficient molding, minimizing damage to cells and demonstrating unique technological advantages and enormous application potential in the engineering construction of tissues and organs. For example, Serpooshan et al. used Faraday waves to drive the rapid aggregation of cardiomyocytes or cardiomyocyte spheroids to form cardiac microtissues. The assembled cells maintained high levels of activity and metabolism, and the resulting cardiac microtissues possessed the contractile and relaxation functions of natural myocardial tissue. Xiao et al. first proposed a multilayer assembly method using Faraday waves, which assembles chambers in situ by stacking layers, using the solidified upper layer as the substrate for the lower layer, thus extending Faraday wave bioassembly technology from single-layer assembly to multilayer assembly. Recently, existing technologies have proposed a multilayer assembly method based on oil-phase assisted filling, reducing the single-layer thickness of multilayer structures and significantly improving the manufacturing precision of Faraday wave multilayer assemblies.

[0003] The aforementioned existing technology has consistently used polymethyl methacrylate (PMMA) as the material for assembling the chambers. Its water droplet contact angle is approximately 80-90°, close to the critical value of 90° between hydrophilic and hydrophobic properties. When liquid is injected into the PMMA chamber, the liquid contacts the chamber sidewalls, forming a gas-liquid-solid three-phase contact line. Due to the combined effects of surface tension and solid surface adhesion, the liquid surface rises at the contact point with the chamber wall, while the middle portion is pulled up to form a concave liquid surface. The contact line is firmly anchored to the top of the sidewall or its vicinity. When the liquid surface fluctuates and attempts to pull the contact line, a strong hysteresis effect occurs, resulting in significant frictional dissipation. This is equivalent to applying a very strong damper to the system. To excite the same Faraday wave pattern as the previous layer, a larger driving amplitude is required. In other words, when the liquid fails to completely fill the chamber, the less liquid is filled, the greater the inhibition of the contact line, the greater the frictional dissipation, and the more difficult it is to assemble and form the pattern. Furthermore, using polymethyl methacrylate as the assembly chamber can affect the integrity of the patterned structure, resulting in blurred edges or no pattern at all, and the pattern in the middle is also affected and deformed. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for fabricating a multilayer acoustic assembly structure based on a hydrophobic chamber. By replacing the polymethyl methacrylate (PMMA) assembly chamber with a more hydrophobic material, this method achieves a minimum single-layer thickness of 0.5 mm, significantly improving the manufacturing precision of the acoustic multilayer assembly.

[0005] To achieve the above objectives, the specific technical solution is as follows.

[0006] This invention provides a method for fabricating an acoustically assembled multilayer structure based on a hydrophobic cavity, comprising the following steps: S1. A photocurable aqueous assembly liquid containing assembly units is added to a hydrophobic chamber, such that the liquid level is below the outer edge of the hydrophobic chamber; the sidewall of the hydrophobic chamber is made of a hydrophobic material; under Faraday wave assembly drive, the assembly units form a specific patterned structure and are then fixed to obtain a cured surface; the parameters of the Faraday wave are as follows: frequency is 10~500Hz, amplitude is 50~1500mV; S2. Repeat the steps in S1 until the solidified surface is consistent with the depth of the hydrophobic cavity to obtain the acoustic assembly multilayer structure. The sidewalls of the hydrophobic chamber are made of a hydrophobic material.

[0007] This invention overcomes the limitations of existing technologies that use polymethyl methacrylate (PMMA) chambers, which require filling the chamber completely, and the need for flipping and demolding to prepare double-layer structures, by using hydrophobic materials as assembly chambers. The preparation method of this invention achieves the preparation of multi-layer structures within a single chamber. Compared with existing technologies that require the stacking or flipping of multiple chambers, this invention effectively simplifies the preparation process, improves preparation efficiency, and produces clear multi-layer structure patterns, ensuring subsequent applications.

[0008] In another preferred embodiment: the hydrophobic material is any one of polytetrafluoroethylene, polystyrene, or polypropylene with a contact angle of 90° to 180°.

[0009] In another preferred embodiment, the depth of the hydrophobic chamber is 0.8 mm to 5 mm.

[0010] In another preferred embodiment, the thickness of each cured surface in the acoustic assembly multilayer structure is 0.5 mm to 3 mm.

[0011] In another preferred embodiment, the assembly unit is any one of nano-iron powder, chitosan powder, or porous microspheres that are insoluble in water and do not chemically react with water; In the photocurable aqueous assembly solution containing the dispersed assembly units, the concentration of the assembly units is 1.5 mg / mL to 25 mg / mL.

[0012] In another preferred embodiment, the photocurable aqueous assembly liquid comprises a photocurable material and an initiator; The photocurable material is any one of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide decellularized matrix, methacrylamide chitosan, methacrylamide carboxymethyl chitosan, methacrylamide sodium alginate, methacrylamide silk fibroin, methacrylamide dextran, methacrylamide chondroitin sulfate, methacrylamide polylysine, acrylamide RGD peptide, polyethylene glycol diacrylate, and polyether F127 diacrylate. The initiator is at least one of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

[0013] In another preferred embodiment, the photocurable aqueous assembly solution contains a photocurable material with a mass-volume concentration of 5% to 15%. In the photocurable aqueous assembly solution, the initiator has a mass-volume concentration of 0.25% to 0.5%.

[0014] In another preferred embodiment, the parameters for photocuring are as follows: illumination time of 30s to 2min, and illumination power of 85mW / cm². 2 .

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention addresses the impact mechanism of the assembly process by proposing a hydrophobic chamber instead of a polymethyl methacrylate (PMMA) chamber. This avoids the limitation that the assembly solution must fill the chamber completely and effectively solves the problems of incomplete oil phase removal and poor biocompatibility caused by using oil-phase-assisted filling to reduce the spacing between adjacent patterns. The hydrophobic chamber eliminates the need for the photocurable aqueous assembly solution to fill the chamber completely. The amount of photocurable aqueous assembly solution can be precisely controlled according to the single-layer thickness, achieving not only precise control of the single-layer thickness but also the fabrication of multilayer structures within the same chamber. The entire process eliminates the need to flip or stack chambers, significantly increasing assembly speed and simplifying the operation. Furthermore, it eliminates the need for oil-phase assistance to reduce interlayer spacing. The method of this invention can achieve a minimum single-layer thickness of 0.5 mm, significantly improving the manufacturing precision of acoustic multilayer assemblies. In addition, the method of this invention can significantly reduce the amount of expensive assembly solutions such as photocurable materials, greatly reducing raw material consumption and manufacturing costs. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the multilayer structure prepared in Example 1.

[0017] Figure 2 This is a schematic diagram of the multilayer structure prepared in Example 2.

[0018] Figure 3 This is a schematic diagram of the multilayer structure prepared in Example 3.

[0019] Figure 4 A is a schematic diagram of the multilayer structure prepared in Example 4; A is a schematic diagram of each layer structure, and B is a schematic diagram of the side structure. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Existing technology discloses a method for constructing a double-layer patterned interface structure, which achieves Faraday wave double-layer assembly through stacked chambers and flipping operations. In this method, the assembly fluid must completely fill the chambers, and the chambers are made of polytetrafluoroethylene (PTFE), mainly utilizing its hydrophobic and non-adhesive properties to facilitate demolding after curing. However, this technology has the following limitations: on the one hand, the stacked chamber method requires the preparation of chambers of different thicknesses, which places high demands on the manufacturing process, resulting in difficulties in controlling layer thickness, low precision, and large interlayer spacing; on the other hand, this method can only obtain a double-layer structure and cannot achieve multi-layer assembly. Although the oil-phase assisted filling method can improve the interlayer spacing problem, it suffers from oil phase residue and poor biocompatibility, limiting its application in the field of tissue engineering.

[0023] To address the aforementioned problems, this invention provides a method for fabricating an acoustic multilayer structure based on a hydrophobic cavity. Unlike existing technologies, this invention utilizes the sidewalls of a hydrophobic material to construct the cavity, with a glass slide at the bottom, eliminating the need for the assembly fluid to completely fill the cavity. The hydrophobic sidewalls effectively reduce the damping effect of liquid surface fluctuations in the incompletely filled state, ensuring the integrity of Faraday waves during multiple assembly processes. This allows for the sequential construction of three or more layers within the same cavity, with each layer's thickness precisely controlled by the amount of assembly fluid used, resulting in small interlayer spacing and high structural precision.

[0024] Compared with existing technologies, the main improvements of this invention are as follows: First, existing technologies utilize the hydrophobicity of PTFE to facilitate demolding after curing, resulting in only a two-layer structure; this invention, however, utilizes hydrophobic sidewalls to reduce the limitation on liquid filling height, enabling in-situ layer-by-layer construction of multi-layer structures without the need for flipping or stacking chambers. Second, in existing technologies, the chamber sidewalls and bottom are both made of PTFE, requiring removal after curing; in this invention, the chamber sidewalls are made of hydrophobic material, and the bottom is a glass slide, eliminating the need for removal after curing. This method not only avoids biocompatibility issues caused by oil phase residue but also significantly improves the manufacturing precision and process controllability of multi-layer structures.

[0025] The following describes in detail an acoustic assembly multilayer structure based on a hydrophobic cavity, its preparation method, and its application. In the examples below, the nano-iron powder was purchased from Maclean's, CAS: 7439-89-6.

[0026] Example 1: A method for fabricating an acoustically assembled multilayer structure based on a hydrophobic cavity, comprising the following steps: S1. Weigh out phenyl (2,4,6-trimethylbenzoyl)lithium phosphate initiator solid powder and dissolve it in phosphate-balanced physiological saline. Heat in a 50°C water bath for 15 minutes, shaking several times during the process to promote dissolution, to obtain an initiator solution with a mass-volume concentration of 0.25%. Subsequently, prepare a photocurable driving solution by weighing out methacrylamide gelatin and dissolving it in the above initiator solution. Heat in a 60°C water bath in the dark for 30 minutes, shaking several times during the process, to obtain a photocurable precursor solution with a mass-volume concentration of 5% methacrylamide gelatin.

[0027] S2. Weigh out the nano-iron powder and add it to the photocurable precursor solution to make the concentration of nano-iron powder reach 1.25 mg / mL. After mixing thoroughly, a photocurable aqueous assembly solution containing nano-iron powder is obtained.

[0028] S3. Add 600 μL of photocurable aqueous assembly liquid containing nano-iron powder to a PTFE assembly chamber with a diameter of 20 mm, a thickness of 3 mm, and a contact angle of 122°. After the nano-iron powder settles to the bottom of the chamber, set the frequency of the acoustic biological assembly instrument to 55 Hz and drive the liquid inside the chamber to vibrate vertically up and down to form a wheel-shaped pattern. Stop the vibration and then irradiate the driving liquid with a 405 nm light source for 2 minutes to fix the assembly structure, obtaining the first cured surface with a thickness of 1.5 mm. Continue adding approximately 600 μL of photocurable aqueous assembly liquid to the chamber. After the iron powder settles to the first cured surface, adjust the acoustic frequency to 55 Hz and drive the assembly to form the same pattern. Then irradiate with a 405 nm light source to cure the methacrylamide gelatin aqueous solution into a gel with a thickness of 1.5 mm, finally obtaining a double-layer wheel-shaped structure. This double-layer wheel-shaped structure is as follows: Figure 1 As shown.

[0029] Example 2: A method for fabricating a multilayer acoustic assembly structure based on a hydrophobic cavity, based on Example 1, with the difference being a standing wave frequency of 72Hz. The specific fabrication process is as follows: S1. Weigh out phenyl (2,4,6-trimethylbenzoyl)lithium phosphate initiator solid powder and dissolve it in phosphate-balanced physiological saline. Heat in a 50°C water bath for 15 minutes, shaking several times during the process to promote dissolution, to obtain an initiator solution with a mass-volume concentration of 0.25%. Subsequently, prepare a photocurable driving solution by weighing out methacrylamide gelatin and dissolving it in the above initiator solution. Heat in a 60°C water bath in the dark for 30 minutes, shaking several times during the process, to obtain a photocurable precursor solution with a mass-volume concentration of 5% methacrylamide gelatin.

[0030] S2. Weigh out the nano-iron powder and add it to the photocurable precursor solution to make the concentration of nano-iron powder reach 1.25 mg / mL. After mixing thoroughly, a photocurable aqueous assembly solution containing nano-iron powder is obtained.

[0031] S3. Add 600 μL of photocurable aqueous assembly liquid containing nano-iron powder to a PTFE assembly chamber with a diameter of 20 mm, a thickness of 3 mm, and a contact angle of 122°. After the nano-iron powder settles to the bottom of the chamber, set the frequency of the acoustic biological assembly instrument to 72 Hz and drive the liquid inside the chamber to vibrate vertically up and down to form a wheel-shaped pattern. Stop the vibration and then irradiate the driving liquid with a 405 nm light source for 2 minutes to solidify the assembly structure, obtaining the first cured surface with a thickness of 1.5 mm. Continue adding approximately 600 μL of photocurable aqueous assembly liquid to the chamber. After the iron powder settles to the first cured surface, adjust the acoustic frequency to 72 Hz and drive the assembly to form the same pattern. Then, irradiate with a 405 nm light source to cure the methacrylamide gelatin aqueous solution into a gel with a thickness of 1.5 mm. Finally, a double-layer wheel-shaped structure is obtained. This double-layer wheel-shaped structure is as follows: Figure 2 As shown.

[0032] Example 3: A method for fabricating a multilayer acoustic assembly structure based on a hydrophobic cavity, based on Example 1, with the difference being a standing wave frequency of 80Hz. The specific fabrication process is as follows: S1. Weigh an appropriate amount of phenyl (2,4,6-trimethylbenzoyl)lithium phosphate initiator solid powder and dissolve it in phosphate-balanced physiological saline. Heat in a 50°C water bath for 15 minutes, shaking several times during the process to promote dissolution, to obtain an initiator solution with a mass-volume concentration of 0.25%. Subsequently, prepare a photocurable driving solution by weighing methacrylamide gelatin and dissolving it in the above initiator solution. Heat in a 60°C water bath in the dark for 30 minutes, shaking several times during the process, to obtain a photocurable precursor solution with a mass-volume concentration of 5% methacrylamide gelatin.

[0033] S2. Weigh out the nano-iron powder and add it to the photocurable precursor solution to make the concentration of nano-iron powder reach 1.25 mg / mL. After mixing thoroughly, a photocurable aqueous assembly solution containing nano-iron powder is obtained.

[0034] S3. Add 600 μL of photocurable aqueous assembly liquid containing nano-iron powder to a PTFE assembly chamber with a diameter of 20 mm, a thickness of 3 mm, and a contact angle of 122°. After the nano-iron powder settles to the bottom of the chamber, set the frequency of the acoustic biological assembly instrument to 80 Hz and drive the liquid inside the chamber to vibrate vertically up and down to form a wheel-shaped pattern. Stop the vibration and then irradiate the driving liquid with a 405 nm light source for 2 minutes to solidify the assembly structure, obtaining the first cured surface with a thickness of 1.5 mm. Continue adding approximately 600 μL of photocurable aqueous assembly liquid to the chamber. After the iron powder settles to the first cured surface, adjust the acoustic frequency to 80 Hz and drive the assembly to form the same pattern. Then, irradiate with a 405 nm light source to cure the methacrylamide gelatin aqueous solution into a gel with a thickness of 1.5 mm. Finally, a double-layer wheel-shaped structure is obtained. This double-layer wheel-shaped structure is as follows: Figure 3As shown.

[0035] Example 4: A method for fabricating a multilayer acoustic assembly structure based on a hydrophobic cavity, based on Example 1, with the difference being that the standing wave frequencies are 80Hz and 75Hz, and the constructed structure is a three-layer structure. The specific fabrication process is as follows: S1. Weigh out solid lithium phenyl (2,4,6-trimethylbenzoyl)phosphate powder, dissolve it in phosphate-balanced physiological saline, and heat in a 40°C water bath for 15 minutes with shaking, to obtain a 0.25% (w / v) lithium phenyl (2,4,6-trimethylbenzoyl)phosphate initiator solution. Then prepare a photocurable driving solution by weighing out methacrylamide gelatin and dissolving it in the above initiator solution, heating in a 60°C water bath in the dark for 20 minutes with shaking, to finally obtain a 5% (w / v) photocurable precursor solution of methacrylamide gelatin.

[0036] S2. Weigh out the nano-iron powder and add it to the photocurable precursor solution to make the nano-iron powder concentration reach 1.25 mg / mL. After mixing thoroughly, the photocurable aqueous phase assembly solution is obtained.

[0037] S3. Add 400 μL of photocurable aqueous assembly liquid containing nano-iron powder to a PTFE assembly chamber with a diameter of 20 mm, a thickness of 3 mm, and a contact angle of 122°. After the nano-iron powder settles to the bottom of the chamber, set the frequency to 80 Hz and drive the liquid in the chamber to vibrate vertically up and down using an acoustic biological assembly instrument until a wheel-shaped pattern is formed. Then stop the vibration and irradiate the curing driving liquid with a 405 nm light source for 2 minutes to fix the assembly structure and obtain the first cured surface with a thickness of 1 mm. Next, add about 400 μL of photocurable aqueous assembly liquid to the chamber, and wait for the iron powder to settle to the bottom. After settling to the first cured surface, the nanoparticles were again assembled using an 80Hz acoustic wave drive to form a wheel-shaped structure. Irradiation with a 405nm wavelength light source cured the methacrylamide gelatin aqueous solution into a gel, resulting in a second cured surface with a thickness of 1mm. Subsequently, 400µL of photocurable aqueous assembly solution was added to the chamber. After the nano-iron powder settled to the second cured surface, it was assembled using a 75Hz drive to form a double-ring pattern. Finally, irradiation with a 405nm wavelength light source cured the methacrylamide gelatin hydrogel, resulting in a third cured surface with a thickness of 1mm. The results are as follows. Figure 4 As shown, during the assembly of the three-layer structure, the added pre-assembly liquid never completely filled the assembly chamber, indicating that the preparation method of this invention can prepare structures with three or more layers, greatly expanding the range of layers.

[0038] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fabricating a multilayer acoustic assembly structure based on a hydrophobic cavity, characterized in that, Includes the following steps: S1. A photocurable aqueous assembly liquid containing assembly units is added to a hydrophobic chamber, so that the liquid level is lower than the outer edge of the hydrophobic chamber; under the Faraday wave assembly drive, the assembly units form a specific patterned structure and are then fixed to obtain a cured surface; the parameters of the Faraday wave are: frequency of 10Hz~500Hz, amplitude of 50mV~1500mV; S2. Repeat the steps in S1 until the solidified surface is consistent with the depth of the hydrophobic cavity to obtain the acoustic assembly multilayer structure. The sidewalls of the hydrophobic chamber are made of a hydrophobic material.

2. The method for fabricating a standing wave multilayer structure based on a hydrophobic cavity according to claim 1, characterized in that, The hydrophobic material is any one of polytetrafluoroethylene, polystyrene, or polypropylene with a contact angle of 90° to 180°.

3. The method for fabricating a standing wave multilayer structure based on a hydrophobic cavity according to claim 1, characterized in that, The depth of the hydrophobic chamber is 0.8mm to 5mm.

4. The method for fabricating a standing wave multilayer structure based on a hydrophobic cavity according to claim 2, characterized in that, The thickness of each cured surface in the acoustic assembly multilayer structure is 0.5mm to 3mm.

5. The method for fabricating a standing wave multilayer structure based on a hydrophobic cavity according to claim 1, characterized in that, The assembly unit is any one of the following: water-insoluble iron nanopowder, chitosan powder, and porous microspheres; In the photocurable aqueous assembly solution containing the dispersed assembly units, the concentration of the assembly units is 0.1 mg / mL to 25 mg / mL.

6. The method for fabricating a multi-layer acoustic assembly structure based on a hydrophobic cavity according to claim 1, characterized in that, The photocurable aqueous assembly solution comprises a photocurable material and an initiator; The photocurable material is any one of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide decellularized matrix, methacrylamide chitosan, methacrylamide carboxymethyl chitosan, methacrylamide sodium alginate, methacrylamide silk fibroin, methacrylamide dextran, methacrylamide chondroitin sulfate, methacrylamide polylysine, acrylamide RGD peptide, polyethylene glycol diacrylate, and polyether F127 diacrylate. The initiator is at least one of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

7. The method for fabricating a multi-layer acoustic assembly structure based on a hydrophobic cavity according to claim 6, characterized in that, In the photocurable aqueous assembly solution, the mass-volume concentration of the photocurable material is 5% to 15%. In the photocurable aqueous assembly solution, the initiator has a mass-volume concentration of 0.25% to 0.5%.

8. The method for fabricating a multilayer acoustic wave assembly structure based on a hydrophobic cavity according to claim 1, characterized in that, The parameters for photocuring are as follows: The light time is 30s~2min, and the light power is 85mW / cm 2 .

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