An ultrasonic response type modular acoustic printing ink, a printing platform and applications thereof

Abstract

This invention discloses an ultrasonic-responsive modular acoustic printing ink, a printing platform, and their applications, relating to the fields of biomedical engineering and biomanufacturing. The ultrasonic-responsive element of this invention is a hollow nanocage composed of fluorinated silk fibroin, and the critical response sound pressure can be set by adjusting the degree of fluorination. The modular acoustic printing ink includes this ultrasonic-responsive element, a biopolymer prepolymer matrix, and optional functional additives. The printing platform integrates a focused ultrasonic transducer, an ink application device, an ultrasonic imaging module, and a control system. This invention uses an ultrasonic triggering method, breaking through the depth limitations of traditional photopolymer printing, to achieve minimally invasive in-situ printing of subcutaneous tissues several centimeters deep. It also features high spatiotemporal resolution, good biocompatibility, and mechanical adaptability, and can flexibly realize functions such as conductivity and drug loading. It is suitable for scenarios such as bioelectrode preparation, visceral wound closure, and controlled drug release, providing a new tool for precision medicine.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and biomanufacturing, and in particular to an ultrasonic-responsive modular acoustic printing ink, a printing platform, and their applications. Background Technology

[0002] 3D bioprinting, as a core technology in the field of biomanufacturing, provides an important tool for constructing biomimetic tissue scaffolds, customized implantable medical devices, and achieving precision medicine by precisely controlling the spatial distribution of biomaterials and cells. It has already demonstrated enormous application potential in biomedical fields such as tissue regeneration, drug screening, and wound repair. Currently, mainstream bioprinting technologies are mainly divided into two categories: one is the in vitro printing-in vivo implantation model, where a three-dimensional structure is first constructed in an in vitro environment using methods such as extrusion or photopolymerization, and then surgically implanted into the target site within the body; the other is in vivo in-situ printing technology, which is still in the exploratory stage and attempts to complete the printing process directly within the biological body to reduce implantation trauma.

[0003] However, existing technologies still face key bottlenecks that are difficult to overcome. For in vitro printing-in vivo implantation, the structure constructed in vitro requires a complex surgical implantation process, which not only causes significant trauma and increases the risk of infection, but may also lead to functional degradation or poor integration with the original tissue due to differences between the in vitro and in vivo physiological environments. Current in vivo in-situ printing technologies mostly rely on photopolymerization, triggering material cross-linking and molding using ultraviolet, visible, or near-infrared light. However, these technologies are limited by the tissue penetration ability of light; biological tissues significantly absorb and scatter light, resulting in an effective printing depth that is typically less than 1 cm, failing to meet the printing needs of deep tissues such as the liver, deep muscles, and lungs. Furthermore, photopolymerization inks often require the addition of photoinitiators, some of which are cytotoxic, and the spatiotemporal accuracy of the photopolymerization process is easily affected by the inhomogeneity of tissue optical properties, making it difficult to achieve precise molding of complex structures.

[0004] Furthermore, the complexity of the internal environment places higher demands on printing technology. Current technologies generally lack the ability to monitor and dynamically control the printing process in real time. Under physiological disturbances such as blood flow and tissue movement, it is difficult to guarantee the spatial accuracy and mechanical stability of the printed structure. At the same time, the mechanical properties of different tissues (such as soft tissue, cartilage, and internal organs) vary significantly. Existing printing materials lack sufficient mechanical adaptability and cannot achieve dynamic mechanical matching with the native tissue, easily leading to implant failure or tissue damage.

[0005] In summary, the limitations of existing bioprinting technologies in terms of deep tissue printing capability, minimal invasiveness, biocompatibility, spatiotemporal accuracy, and mechanical adaptability have become technical obstacles restricting their application in complex clinical scenarios. Therefore, developing an in vivo in-situ 3D printing technology that can penetrate deep tissues, achieve minimally invasive operations, and possess excellent biocompatibility and precise controllability has become a key issue that urgently needs to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an ultrasonic-responsive modular acoustic printing ink, a printing platform, and its applications to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of the present invention is to provide an ultrasonic response element (URE), wherein the ultrasonic response element is a hollow nanocage composed of fluorinated silk fibroin.

[0009] Furthermore, the degree of fluorination of the fluorinated silk fibroin is 20-40%. Here, the degree of fluorination is the percentage of the mass of the fluoride relative to the mass of the grafted polymer.

[0010] The ultrasonic response element of the present invention can directly respond to the mechanical force of ultrasonic waves to undergo structural transformation, and its critical response sound pressure is 0.6 MPa to 4.4 MPa.

[0011] The ultrasonic response element of the present invention can set a specific critical response sound pressure by adjusting the degree of fluorination of fluorinated silk fibroin.

[0012] Furthermore, the surface of the hollow nanocage is modified with a dopamine molecular layer.

[0013] The second technical solution of the present invention provides a method for preparing the above-mentioned ultrasonic response element, comprising the following steps:

[0014] (1) The silk fibroin solution was mixed with a fluorine-containing reagent and reacted to obtain fluorinated silk fibroin;

[0015] (2) The fluorinated silk fibroin is dispersed in a microemulsion to form a nanocage precursor with the emulsion droplets as templates;

[0016] (3) The nanocage precursor is solidified and the template is removed to obtain the hollow nanocage.

[0017] If it is necessary to modify the dopamine molecular layer, disperse the hollow nanocages obtained in step (3) in the dopamine solution, and separate and purify them after the reaction.

[0018] The third technical solution of the present invention provides a modular acoustic printing ink (US-Ink) comprising the above-mentioned ultrasonic response element and a biopolymer prepolymer matrix; wherein the ultrasonic response element is dispersed in the biopolymer prepolymer matrix.

[0019] Furthermore, the biopolymer prepolymer matrix contains thiol- and azide-modified hyaluronic acid and thiolized polylysine.

[0020] Furthermore, the modular acoustic printing ink also contains functional additives.

[0021] The functional additive is selected from one or more of conductive materials, therapeutic drugs, growth factors, or imaging agents; the conductive materials include PEDOT:PSS or carbon nanotubes.

[0022] The fourth technical solution of the present invention provides the application of the above-mentioned modular acoustic printing ink in the preparation of in vivo bioelectrodes, visceral (including liver or lung) wound sealing materials, or in vivo drug controlled release reservoirs.

[0023] Fifth technical solution of the present invention: providing an in vivo deep tissue acoustic printing platform, comprising:

[0024] Transducer devices used to generate focused ultrasound;

[0025] A device for containing and applying the aforementioned modular acoustic printing ink;

[0026] An ultrasound imaging module for imaging the interior of an organism and guiding the transducer device; for real-time positioning of the target region within the organism and monitoring the printing process.

[0027] A control system for controlling the three-dimensional motion and parameter adjustment of the transducer device; used to control the spatial position of the transducer device.

[0028] Furthermore, the in vivo deep tissue acoustic printing platform of the present invention also includes an intelligent control system for parsing the printing path code and controlling the output sound pressure of the transducer device and the movement parameters of the platform.

[0029] The sixth technical solution of the present invention provides a method for in-situ 3D printing in deep tissues of a living organism, using the above-mentioned modular acoustic printing ink (US-Ink) and utilizing the above-mentioned in vivo deep tissue acoustic printing platform, by using focused ultrasound to trigger an ultrasound response element (URE) to cause the modular acoustic printing ink to cross-link and form at the target tissue location.

[0030] The more specific steps are as follows:

[0031] (1) Modular acoustic printing ink is delivered to the target deep tissue area in the body via percutaneous puncture or minimally invasive intervention;

[0032] (2) Using an in vivo deep tissue acoustic printing platform (integrated ultrasound printing system), the target area is located by ultrasound imaging guidance module;

[0033] (3) Activate the transducer device that generates focused ultrasound, emit ultrasound with a preset sound pressure, trigger the ultrasonic response element in the modular acoustic printing ink to undergo structural transformation, and cause the prepolymer matrix to crosslink to form a preset 3D structure.

[0034] (4) The molding process is monitored in real time by the ultrasonic imaging guidance module to complete the printing.

[0035] The preset sound pressure in step (3) is 0.6 MPa to 4.4 MPa, and matches the critical response sound pressure of the ultrasonic response element.

[0036] This invention employs a collaborative design of ultrasonic response elements, modular inks, and an integrated system. Through an innovative triggering mechanism and system integration, it achieves precise and controllable printing of deep tissues within the body.

[0037] The core of this platform lies in the organic integration of three key components. Among them, the Ultrasonic Response Element (URE) is the core triggering unit of the entire technical solution. It is a hollow nanocage structure constructed from fluorinated silk fibroin using a microemulsion template method, and its surface can be modified with dopamine functional molecules to optimize interfacial properties. This invention's URE does not rely on traditional thermal or cavitation effects, but directly responds to the mechanical force of ultrasound, undergoing structural transformation or rupture, thereby triggering subsequent cross-linking reactions. More importantly, it possesses unique programmability. By controlling the degree of fluorination, elements with different critical response sound pressures can be fabricated. Combined with controllable programming of sound pressure / power, selective cross-linking can be achieved, laying the foundation for acoustic programming printing of complex patterns, mechanical gradients, and heterogeneous structures in vivo.

[0038] In this invention, modular acoustic printing ink (US-Ink) serves as the functional carrier for printing. It uses biocompatible polymers containing complementary reactive groups such as thiol and azide (e.g., hyaluronic acid, polylysine) as the prepolymer matrix, with URE (urea-formaldehyde resin) uniformly dispersed within it as an ultrasonically triggered "crosslinking switch." This ink system employs a modular design, allowing for the flexible incorporation of conductive materials (e.g., PEDOT:PSS, carbon nanotubes), therapeutic drugs, growth factors, or developing agents according to application requirements. This achieves "plug-and-play" functionality, greatly enhancing the flexibility and versatility of applications.

[0039] The integrated ultrasonic printing system of this invention provides precise hardware support for the entire printing process, including a transducer array that can generate controllable focused ultrasonic waves (adapting to different printing depths, precision, and mechanical requirements), a three-dimensional precision displacement platform that controls the spatial position of the transducers (enabling complex 3D path printing), an ultrasonic imaging module that guides positioning and monitors the process in real time, and an intelligent control system that integrates hardware control, G-code parsing, and real-time parameter adjustment, ensuring the precision and controllability of the printing process.

[0040] The core design of this invention achieves breakthroughs in the following aspects:

[0041] (1) Breaking through the bottleneck of deep printing, the strong penetrability of focused ultrasound is used to effectively act on deep tissues several centimeters below the skin, overcoming the depth limitation of traditional photopolymerization printing;

[0042] (2) Achieve high-precision controllable molding. Through real-time guidance of ultrasonic imaging and programmable sound pressure, the position, sequence and degree of crosslinking can be precisely controlled, achieving a high spatiotemporal resolution of 200 μm linewidth in a complex internal environment.

[0043] (3) Improve biocompatibility and mechanical compatibility. The core materials (silk fibroin, hyaluronic acid, etc.) have excellent biocompatibility and can be customized to match the mechanical properties of different tissues by selecting different sound pressure sensitive UREs or adjusting the ink composition, so as to achieve "in-situ integration".

[0044] (4) It enables modularization of functions and diversification of applications. Through simple mixing, it can achieve functions such as conductivity, drug loading, and imaging, thus expanding its application in fields such as bioelectronics, wound repair, and controlled drug release.

[0045] (5) It enables minimally invasive treatment. The printing process can be completed through percutaneous puncture or minimally invasive intervention. Combined with ultrasound imaging, it enables in-situ real-time monitoring, providing an innovative tool for precision medicine.

[0046] The printing process can be completed through percutaneous puncture, and with real-time ultrasound monitoring, it effectively reduces surgical trauma and infection risks. It successfully solves the technical difficulties of existing technologies in deep tissue printing, biocompatibility, spatiotemporal accuracy and mechanical adaptability, and provides a brand-new technical tool for precision medicine.

[0047] The present invention discloses the following technical effects:

[0048] This invention employs ultrasonic triggering technology, overcoming the depth limitations of traditional photopolymerization printing. It can precisely target deep tissues several centimeters below the skin, enabling in-situ printing under minimally invasive conditions. By utilizing the programmable acoustic pressure characteristics of the ultrasonic response element and combining it with the real-time guidance function of ultrasonic imaging, high spatiotemporal resolution printing is achieved, ensuring the precise formation of complex three-dimensional structures in vivo. By adjusting the fluorination degree of the ultrasonic response element or optimizing the composition of the printing ink, the mechanical properties of different tissues can be customized, significantly improving the integration effect between the implant and the native tissue.

[0049] The modular ink design of this invention supports "plug-and-play" functions such as conductivity, drug loading, and imaging, greatly expanding its application scope in multiple scenarios such as bioelectrode preparation, visceral wound sealing, and drug controlled-release storage construction. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is a schematic diagram of the fabrication process of an ultrasonic response element (URE).

[0052] Figure 2 Scanning electron microscope (a), transmission electron microscope (b), and particle size distribution diagram (c) of the ultrasonic response element (URE).

[0053] Figure 3 Ultrasonic response characterization diagrams of ultrasonic response elements (UREs) (a) Transmission electron micrographs of UREs without FUS treatment and with FUS treatment; b) Scanning transmission electron micrographs of UREs with FUS treatment; c) Particle size distribution curves of UREs after FUS treatment at different times.

[0054] Figure 4 Characterization diagrams of the critical response sound pressure of ultrasonic response elements (URE-1, URE-2, URE-3) with different degrees of fluorination;

[0055] Figure 5 The rheological test results of modular acoustic printing ink (US-Ink) with URE percentage content are shown in the figure.

[0056] Figure 6The images show the in vitro printing performance of the acoustic printing platform (A is the printing result of a porous scaffold with a complex spatial structure, and B is the printing result of a large-volume patterned scaffold); where I is the actual printed image of the porous scaffold, II and III are the front views of the porous scaffold, IV, V, and VI are the top views of the porous scaffold, and VII and VIII are the SEM images of the porous scaffold.

[0057] Figure 7 The diagram shows the construction and validation of the in vivo deep tissue printing platform; a) is a schematic diagram of the system device and the in vivo printing process; b) shows the ink focusing ultrasound response and cross-linking process; c) is a comparison of ultrasound images of the target tissue before and after printing; d) is an anatomical evaluation diagram of the printing site.

[0058] Figure 8 The images show the electrophysiological signal monitoring results of the in vivo printed bioelectrode of this invention and a comparison with commercial electrodes; a) ECG and EMG signal acquisition spectra of the in vivo printed bioelectrode of this invention; b) ECG signal spectra of the in vivo printed bioelectrode of this invention 24 hours later; c) ECG signal spectra of the commercial electrode 24 hours later.

[0059] Figure 9 This is a diagram showing the experimental results of in vivo acoustic printing sutures according to the present invention. Detailed Implementation

[0060] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0061] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0062] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0063] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0064] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0065] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0066] Example 1: Fabrication of an Ultrasonic Response Element (URE)

[0067] The experimental materials used in this embodiment are: silk fibroin, heptafluorobutyric anhydride, tridecafluoroheptanoic acid, EDC, NHS, sodium carbonate, hyaluronic acid, polylysine, ethanol, PEDOT:PSS, dopamine hydrochloride, sodium bis(2-ethylhexyl)sulfosuccinate (AOT), isooctane, lithium bromide, sodium hydroxide, carbon nanotubes, graphene, DMSO, PEG-SH, PEG-CO, and PEG-N3.

[0068] The preparation steps are as follows:

[0069] (1) Preparation of regenerated silk fibroin (RSF): The raw silk from the silkworm cocoon was chopped and boiled three times (30 minutes each time) in 0.02M Na2CO3 aqueous solution. It was then washed with deionized water several times to completely remove the sericin. Subsequently, the dried degummed silk was dissolved in 9M lithium bromide (LiBr) solution at 60℃ for 2 hours. After filtration, the obtained silk fibroin (SF) solution was placed in a cellulose bag (MWCO 1400 Da) and dialyzed with distilled water at room temperature for at least 3 days to remove Na2CO3 and lithium bromide. Finally, the obtained regenerated silk fibroin solution was freeze-dried for later use.

[0070] (2) Preparation of fluorinated silk fibroin: Dissolve regenerated silk fibroin in deionized water to maintain a concentration of 3%wt and hydrate thoroughly overnight; dissolve heptafluorobutyric anhydride in DMSO (concentration 10 mg / mL), and add 5 mL of this solution dropwise to the silk fibroin aqueous solution (heptafluorobutyric anhydride: silk fibroin volume ratio of 1:5). After vigorous vortexing for 2 minutes, adjust the pH of the reaction system to 8.0 in real time with 1N sodium hydroxide aqueous solution. After reacting for 6 hours, place it in a refrigerator at 4℃ overnight; then pour the reaction solution into a dialysis bag, dialyze with distilled water at room temperature for 3 days, and freeze dry for later use.

[0071] (3) Preparation of two-phase microemulsion: Add biocompatible surfactant AOT to 1% isooctane and stir until completely dissolved (mass ratio of AOT to isooctane 1:1); add 5 mL of fluorinated silk fibroin solution with a concentration of 15 mg / mL, adjust pH to 6.8, and mix thoroughly with an ultrasonic mixer for 30 minutes to obtain fluorinated silk fibroin microemulsion (FSF RME), which is stored at 4℃ for later use.

[0072] (4) Weaving of nanocages: Sodium carbonate nanoparticles were first prepared as templates and suspended in an aqueous solution (concentration 0.5 mg / mL) by ultrasonication and vortexing. FSF RME was added to the template solution at a volume ratio of 1:5 and vortexed vigorously until the mixed solution separated into layers. The upper oil phase was discarded, and the lower milky white emulsion was vortexed again in an ice bath for 10 minutes. The silk fibroin-based nanocages (NC) were obtained by centrifugation at 11,000 rpm for 20 minutes. After washing with water 3 times, the nanocages were stored at -20℃. Then, a pH 5.0 PBS solution was prepared as an etching solution, and a crosslinking agent was added. The nanocages were resuspended in the solution (concentration 2% wt) and etched by stirring at 4℃ for 2 hours. The nanocages were collected by centrifugation at 11,000 rpm for 20 minutes again, washed with water 3 times to remove calcium ions, and then lyophilized and stored at -20℃.

[0073] (5) Nanocage surface modification: The nanocages were resuspended in deionized water (concentration 1% wt); EDC / NHS solution (concentration 5 mg / mL, mass ratio 1:1) was prepared, and the nanocage suspension was mixed with the EDC / NHS solution in equal volume and stirred at room temperature for 6 hours; 2 mg of dopamine was added, and the mixture was vortexed vigorously for 2 minutes. The pH of the reaction system was adjusted to 6.0 in real time with hydrochloric acid. After reacting for 6 hours, the mixture was placed in a 4℃ refrigerator overnight; the reaction solution was poured into a dialysis bag and dialyzed with distilled water at room temperature for 3 days. After lyophilization, the surface dopamine-modified ultrasonic response element (URE) was obtained.

[0074] The prepared URE was characterized, and the results are as follows:

[0075] Morphological characterization: Scanning electron microscopy and transmission electron microscopy revealed that the prepared URE exhibited a regular spherical hollow cage-like structure with a particle size of approximately 150 nm and a uniform particle size distribution (as shown in Figure 1). Figure 2 (as shown)

[0076] Ultrasonic responsiveness: Figure 3The images show the ultrasonic responsiveness characterization of the ultrasonic response element (URE); a) transmission electron microscopy (TEM) images of the URE before and after FUS treatment; b) scanning TEM image of the URE after FUS treatment; the FUS treatment conditions were 1.1 MHz, 2 W / cm², 10 s. The TEM results confirmed the collapse of the nanocage structure, indicating its rapid mechanical force response capability; the particle size distribution of the URE after ultrasonic treatment (1.1 MHz, 2 W / cm²) at different times (5 s, 10 s) is shown below. Figure 3 As shown in c.

[0077] Sound pressure sensitivity: The critical response sound pressures of three UREs with different degrees of fluorination (URE-1, URE-2, and URE-3) were 0.6 MPa, 2.0 MPa, and 4.4 MPa, respectively, confirming their programmable sound pressure sensitivity (e.g., sound pressure sensitivity). Figure 4 (As shown). Among them, the fluorination degrees of URE-1, URE-2, and URE-3 are 39%, 28%, and 20%, respectively.

[0078] Example 2: Preparation of Modular Acoustic Printing Ink (US-Ink)

[0079] The experimental materials used in this embodiment are: hyaluronic acid, EDC, NHS, NH2-PEG-SH, NH2-PEG-N3, polylysine, COOH-PEG-SH, deionized water, hydrochloric acid, ultrasonic response elements (URE-1, URE-2, URE-3 prepared in Example 1), conductive additives (PEDOT:PSS, graphene, carbon nanotubes (CNTs), silver nanowires, MXene), and oil-based dyes.

[0080] (1) Preparation of thiol-azide modified hyaluronic acid (SH-HA-N3): Hyaluronic acid was dissolved in deionized water to maintain a concentration of 10% wt. At the same time, an EDC / NHS solution (dissolved in deionized water, final concentration 10 mg / mL, mass ratio of EDC to NHS 1:1) was prepared. The hyaluronic acid solution and the EDC / NHS solution were mixed in equal volumes and stirred at room temperature for 6 hours. Then, 2 mg of NH2-PEG-SH and NH2-PEG-N3 were added respectively, and the mixture was vortexed vigorously for 2 minutes. The pH of the reaction system was adjusted to 6.0 in real time with hydrochloric acid. After reacting for at least 6 hours, the mixture was placed in a refrigerator at 4°C overnight. The solution after complete reaction was poured into a dialysis bag and dialyzed with distilled water at room temperature for 3 days. After lyophilization, thiol-azide modified hyaluronic acid was obtained.

[0081] (2) Preparation of thiolated polylysine (SH-PLL): First, COOH-PEG-SH was fully dissolved in deionized water to a concentration of 5 mg / mL. Then, an EDC / NHS solution of the same specification was prepared. The COOH-PEG-SH solution and the EDC / NHS solution were mixed in equal volumes and stirred at room temperature for 6 hours. Then, an equal volume of the mixture was added to a 5% wt polylysine aqueous solution and vortexed vigorously for 2 minutes. The pH of the reaction system was adjusted to 6.0 in real time with hydrochloric acid. After reacting for 6 hours, the mixture was placed in a refrigerator at 4°C overnight. The reaction solution was poured into a dialysis bag and dialyzed with distilled water at room temperature for 3 days. After freeze-drying, thiolated polylysine was obtained.

[0082] (3) Preparation of injectable bio-ink: The ultrasonic-responsive smart hydrogel prepolymer prepared in this invention, called US ink, is prepared by mixing a matrix composed of thiol-azide-modified hyaluronic acid, thiol-modified polylysine, and three ultrasonic response elements. First, 2 wt.% thiol-azide-modified hyaluronic acid and 5 wt.% thiol-modified polylysine are mixed and vacuum-treated to form a prepolymer hydrogel matrix. Then, the three ultrasonic response elements URE-1, URE-2, and URE-3 are blended with the hydrogel prepolymer matrix to finally form ultrasonic printing biosafety ink (US-Ink). The amount of ultrasonic response elements added and the grafting / blending method are optimized based on subsequent rheological tests and crosslinking mechanism characterization.

[0083] Functional doping can also be performed on this basis, and adjustments can be made according to application requirements. For example, to prepare conductive ultrasonic printing bio-ink, 5 wt.% of conductive additives (such as PEDOT:PSS, graphene, carbon nanotubes, silver nanowires or MXene) can be added to US-Ink. If further conductivity is required, an additional 0.2 wt.% of silver nanowires can be added to the conductive US ink containing carbon nanotube additives. In addition, to enhance the visualization effect during the printing process, 0.2 wt.% of oily dyes can be added to the basic prepolymer.

[0084] The performance of the US-Ink (without additives) prepared in Example 2 was tested, and the results are as follows:

[0085] Rheological test:

[0086] Rheological properties were characterized using an MCR 302 rheometer (Antonpah, Austria) equipped with an 8 mm diameter parallel plate measurement system. 75 μL of US-Ink was used for testing, with a gap size of 0.5 mm. First, to evaluate the crosslinking behavior of US-Ink in response to ultrasound, the storage modulus (G') and loss modulus (G'') of the pre-gelled solution were monitored. US-Ink was first equilibrated at 37°C for 15 min, then treated with FUS (1 MHz), while G' and G'' were recorded at 10% strain amplitude and 1 Hz frequency. The gelation time was determined as the intersection of the G' and G'' curves.

[0087] Figure 5 The figure shows the rheological test results of modular acoustic printing ink (US-Ink) with different URE percentage contents; G' is the storage modulus and G'' is the dissipation modulus. US-Ink exhibits shear-thinning behavior, which makes it more suitable for injection delivery via minimally invasive methods; under simulated ultrasonic triggering conditions (1.5 MPa), its storage modulus (G') rapidly increases from 10 Pa to 6500 Pa within 30 seconds, enabling the rapid formation of a stable hydrogel.

[0088] In vitro printing test: Using the constructed printing system in a simulated tissue (agarose gel) environment, complex patterns (such as grids and spirals) with a line width of 200 μm were successfully printed, proving that the ink has high-resolution printing capabilities.

[0089] Figure 6 The images show the in vitro printing performance of the acoustic printing platform (A is the printing result of a porous scaffold with a complex spatial structure, and B is the printing result of a large-volume patterned scaffold). Among them, I is the actual printed image of the porous scaffold, II and III are the front views of the porous scaffold (II has a large porosity, and III has a small porosity), IV, V, and VI are the top views of the porous scaffold (IV, V, and VI have porosities from large to small in sequence), and VII and VIII are the SEM images of the porous scaffold.

[0090] Example 3: Construction of an Ultrasonic Printing System

[0091] The in vivo acoustic printing system constructed in this embodiment is an integrated platform adapted to the ultrasonic response element (URE) prepared in Example 1 and the modular acoustic printing ink (US-Ink) prepared in Example 2. The specific assembly scheme is as follows:

[0092] The core voice control unit of the system uses a single-element FUS transducer (model H-102, H-108, brand SonicConcepts), with operating frequencies covering 1.1 MHz, 2.65 MHz, 8.75 MHz and 12.86 MHz. This wide frequency range matches the ultrasonic response characteristics of the URE in Example 1, and the wide frequency design ensures accurate matching of the triggering conditions of various UREs.

[0093] To further calibrate the focus of the ultrasonic energy and ensure that the ultrasonic energy is accurately applied to the URE in the US-Ink, a short electrical pulse was initially generated by exciting the transducer using a Panametrics 5072PR pulse receiver. At the same time, the received signal was monitored using an oscilloscope (Rigol DS1054, UltraVision Tech.). The focus was located through signal analysis to ensure the accuracy of ultrasonic triggering.

[0094] The transducer is fixedly mounted on an electric positioning system, which enables high-precision three-dimensional spatial control and adjustable movement speed. Its design is adapted to the characteristics of US-Ink in Example 2. US-Ink in Example 2 exhibits shear-thinning properties and requires minimally invasive delivery. The three-dimensional precision displacement platform can be coordinated with the delivery path to achieve precise printing of complex 3D patterns. Simultaneously, the adjustable movement speed matches the ultrasonic crosslinking rate of US-Ink, preventing incomplete molding due to excessive speed or material diffusion due to excessively slow movement. The transducer is driven by a function generator (model AFG3252, brand Tektronix, made in Beaverton, OR). Its output signal is amplified by an RF power amplifier (model A075, brand Electronics and Innovation, made in Rochester, NY) before being transmitted to the transducer. The output power can be displayed in real time via the amplifier, and the power adjustment range perfectly matches the critical response sound pressure of the URE in Example 1 (0.6, 2.0, 4.4 MPa), ensuring selective triggering of the URE through power regulation. For high-frequency operating modes of 8.75 MHz and 12.86 MHz, the RF power amplifier is connected to the FUS transducer through an access matching network to optimize signal transmission efficiency and power stability, ensuring that the URE can still respond stably at high frequencies, thereby ensuring rapid cross-linking and molding of US-Ink.

[0095] The system's motion control follows G-code instructions. The transducer moves along a path planned by the preset G-code, coordinating with the ultrasonic triggering sequence to achieve the target printed pattern. Key parameters during the printing process, such as ultrasonic power (matching URE response requirements) and transducer moving speed (matching US-Ink molding rate), can be preset in advance or adjusted in real time through programming. This ensures that the US-Ink of Example 2 (including the URE of Example 1) can accurately complete in-situ 3D printing of deep tissues in vivo under the control of this system (e.g., Figure 7 ).

[0096] Example 4: Customized applications of in vivo acoustic printing (animal experiments)

[0097] This embodiment uses the ultrasonic response element (URE) prepared in Example 1, the modular acoustic printing ink (US-Ink) prepared in Example 2, and the focused ultrasound printing system built in Example 3 to conduct animal experiments:

[0098] 1. Subcutaneous bioelectrode printing and electrophysiological signal detection

[0099] Conductive US-Ink containing carbon nanotube (CNT) additives prepared in Example 2 (CNT addition percentage of 10%) was used to print bioelectrodes subcutaneously in animals using the focused ultrasound printing system of Example 3 (focused ultrasound frequency: 1MHz, power: 5W, sound pressure controlled between 4-4.4 MPa; printing speed: 5 mm / min; printing tank temperature controlled at 37℃; G-Mode was used to control the printing probe, and 16-channel bioelectrodes were printed). These bioelectrodes were then used for electrocardiogram (ECG) and electromyography (EMG) signal monitoring.

[0100] In the experiment, the reference electrode was fixed to the animal's leg; during ECG monitoring, the printed working electrodes were placed on the animal's right and left arms, respectively; during EMG monitoring, the working electrodes were attached to both ends of the animal's biceps brachii. Electrophysiological signals were captured using an open-source hardware jammer (model SparkFun AD8232) and subsequently low-pass filtered at 50 Hz using MATLAB software to eliminate interference signals.

[0101] Figure 8 This document presents the monitoring results of electrocardiogram (ECG) and electromyography (EMG) signals by the in vivo printed bioelectrode of this invention, and a comparison with commercial electrodes; a) shows the acquisition results of ECG and EMG signals by the subcutaneous acoustic printed bioelectrode; b) shows the changes in ECG signal spectrum after 24 hours of use of the subcutaneous printed electrode; c) shows the changes in ECG signal peak spectrum (significantly weakened) after 24 hours of use of the commercial wet electrode.

[0102] like Figure 8 As shown, compared with commercial electrodes, the subcutaneous bioelectrode printed in this invention recorded ECG signals with a comparable signal-to-noise ratio, and key waveform features such as the P wave, QRS complex, and T wave were clearly distinguishable. Thanks to the excellent biocompatibility of US-Ink with in vivo tissues, the printed electrode adhered tightly to the subcutaneous tissue, and the signal baseline remained stable throughout the animal's activity. Comparative tests showed that after 24 hours of use, the signal intensity of the commercial electrode (wet electrode) significantly decreased, while the printed electrode of this invention maintained stable signal output within the same timeframe, demonstrating superior tissue integration and long-term stability.

[0103] 2. Printing of internal organ wound closure

[0104] Rats were selected as experimental subjects, and a standardized incision of approximately 5 mm was created on the surface of their livers to simulate an internal organ trauma scenario. The basic US-Ink (without functional additives) prepared in Example 2 was drop-coated onto the wound area. Subsequently, the focused ultrasound printing system of Example 3 was activated, and an ultrasound signal at a frequency of 1.1 MHz and a sound pressure level of 2.0 MPa was used to scan the wound, triggering a structural transformation of the URE in the US-Ink, which caused the ink to rapidly cross-link and form a hydrogel sealing layer. A comparative example was set up: a prepolymer matrix without the URE prepared in Example 1, but with all other components identical, was used to treat rat liver wounds under the same ultrasound parameters.

[0105] Figure 9 This image shows the results of an in vivo acoustic printing suture experiment, demonstrating the repair effects in a liver injury bleeding model and a lung perforation model. Upon ultrasound triggering, the URE-containing base US-Ink rapidly cross-links to form a dense hydrogel. This hydrogel adheres tightly to the liver tissue surface, successfully stopping wound bleeding and achieving effective sealing. In the comparative example, the prepolymer matrix without URE failed to undergo effective cross-linking under the same ultrasound conditions, forming only a fluid liquid and failing to form a stable sealing layer. The wound continued to bleed, resulting in sealing failure. These results clearly demonstrate the crucial role of URE in triggering in-situ cross-linking of US-Ink in Example 1 and also verify the feasibility of the entire printing scheme in visceral wound closure scenarios.

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An ultrasonic response element, characterized in that, The ultrasonic response element is a hollow nanocage made of fluorinated silk fibroin.

2. The ultrasonic response element according to claim 1, characterized in that, The fluorinated silk fibroin has a fluorination degree of 20-40%.

3. The ultrasonic response element according to claim 1, characterized in that, The hollow nanocage is surface-modified with a dopamine molecule layer.

4. A modular acoustic printing ink, characterized in that, It comprises an ultrasonic response element as described in any one of claims 1-3 and a prepolymer matrix; the ultrasonic response element is dispersed in the prepolymer matrix.

5. The modular acoustic printing ink according to claim 4, characterized in that, The prepolymer matrix contains thiol- and azide-modified hyaluronic acid and thiolized polylysine.

6. The modular acoustic printing ink according to claim 4, characterized in that, It also contains functional additives; the functional additives are selected from one or more of conductive materials, therapeutic drugs, growth factors or contrast agents; the conductive materials include PEDOT:PSS or carbon nanotubes.

7. The application of the modular acoustic printing ink as described in any one of claims 4-6 in the preparation of in vivo bioelectrodes, visceral wound sealing materials, or in vivo controlled-release drug reservoirs.

8. An in vivo deep tissue acoustic printing platform, characterized in that, include: Transducer devices used to generate focused ultrasound; A means for containing and applying the modular acoustic printing ink as described in any one of claims 4-6; An ultrasound imaging module used to image the interior of an organism and guide the transducer device. A control system for controlling the three-dimensional motion and parameter adjustment of the transducer device.

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