An ultrasonic nanobubble coupling agent, its preparation method and application

Abstract

This invention provides an ultrasonic nanobubble coupling agent, its preparation method, and its application. The invention uses Cel@PFH@PLGA-NH2 nanobubbles prepared by a double emulsification method and aminoated hyaluronic acid as raw materials. A novel ultrasonic nanobubble coupling agent is prepared through Schiff base reaction and dynamic covalent cross-linking of aldehyde-based hyaluronic acid. This ultrasonic nanobubble coupling agent can effectively transfer drugs across the skin's dual barriers and deliver them to deeper layers by amplifying the cavitation effect of ultrasound, achieving a transdermal depth of 728 μm, significantly improving transdermal depth and penetration efficiency. Furthermore, this ultrasonic nanobubble coupling agent also possesses excellent adhesion and self-healing properties, enabling it to effectively restore its original state.

Description

Technical Field

[0001] This invention belongs to the field of ultrasonic coupling agent technology, specifically relating to an ultrasonic nanobubble coupling agent, its preparation method, and its application. Background Technology

[0002] Non-invasive drug delivery is a method of drug administration that delivers medication directly and effectively into the body through the skin, mouth, nose, anus, rectum, etc., without involving surgery or injection incisions. Compared with traditional drug delivery methods, non-invasive drug delivery is safer, more convenient, painless, and carries no risk of infection. Therefore, it is suitable for the treatment of certain special populations (such as infants and the elderly) and specific diseases. In addition, non-invasive drug delivery improves patient compliance and treatment outcomes and can prevent the occurrence of complications.

[0003] While non-invasive drug delivery technologies have benefited more patients, several challenges remain. First, some drugs, when passing through the skin or nasal mucosa, are hindered by the stratum corneum and basement membrane, preventing them from penetrating deep into tissues and resulting in suboptimal therapeutic effects. Second, non-invasive drug delivery requires a longer management time; compared to injections, this method often takes longer to achieve therapeutic effects due to the slow, sustained release of the drug. Third, dosage control is difficult; since non-invasive drug delivery is typically administered via patches and sprays, a stable drug supply cannot be guaranteed, thus affecting efficacy. Therefore, addressing the shortcomings of existing non-invasive drug delivery methods necessitates the development of a more effective, controllable, and deep-tissue-delivery non-invasive drug delivery method.

[0004] The skin, one of the largest organs in the human body, is composed of multiple layers, including the epidermis, basement membrane, dermis, and subcutaneous tissue. The stratum corneum (SC), composed of 15-20 micrometers thick layers of dead skin cells filled with keratin, has a "brick-and-mortar" structure and is the primary barrier for transdermal drug penetration. Furthermore, the basement membrane, a non-cellular layer between the dermis and epidermis, provides support, connects the two layers, and regulates cell migration, proliferation, and tissue regeneration. In addition, the skin's basement membrane filters out substances that penetrate the skin, making it difficult for larger molecular weight, highly polar, and hydrophobic drugs to penetrate and reach the dermis. Therefore, transdermal drug delivery for non-invasive purposes is often less effective. Only transdermal drug delivery methods that can simultaneously open both the stratum corneum and basement membrane barriers can truly achieve efficient, non-invasive, and deep dermal drug delivery.

[0005] To date, researchers have used chemical or physical methods to increase drug permeability across the skin barrier. Commonly used chemical methods primarily involve permeation enhancers such as dimethyl sulfoxide (DMSO) and propylene glycol (PG). However, even with the aid of chemical permeation enhancers, basement membrane penetration has not been sufficiently effective. In contrast, the most widely accepted physical method for microneedle drug delivery involves using tiny needles to penetrate the skin, improving drug delivery efficiency through invasiveness. However, this invasive method easily leads to local skin barrier disruption and stratum corneum shedding. Furthermore, the application of traditional thermal ablation and electroporation is limited due to wound formation and safety concerns. Therefore, a truly non-invasive and highly efficient deep tissue drug delivery method remains lacking.

[0006] Ultrasound, also known as sonophoresis, not only has the advantages of being non-invasive, economical, and convenient, but also allows for precise spatiotemporal control of drug delivery. The cavitation effect of ultrasound allows drugs to non-invasively penetrate the skin barrier and enter the dermis, where they are subsequently absorbed by the body's circulation. The *Chinese Journal of Medical Ultrasound (Electronic Edition)* (2016) reported on the progress of low-frequency ultrasound transdermal drug delivery, describing it as a non-invasive transdermal drug delivery method that can promote drug penetration under ultrasound. Low-frequency ultrasound-enhanced penetration can more effectively improve the permeability of the skin, especially the stratum corneum, making passive drug diffusion more rapid and enabling transdermal enhanced penetration of large molecule drugs.

[0007] Low-frequency ultrasound-enhanced transdermal drug delivery offers unique advantages over traditional methods due to its painless, non-invasive, highly effective, and first-barrier-free nature. However, current ultrasound-enhanced methods still suffer from shallow transdermal depths, failing to effectively penetrate both skin barriers, and prolonged transdermal drug delivery time.

[0008] Ultrasound coupling agents are medical products composed of aqueous polymer gels that serve as intermediate media for the propagation of sound waves, essential for ultrasound to achieve its therapeutic and diagnostic effects. Researchers have conducted extensive research on ultrasound coupling agents to enhance their permeability, but current capabilities remain limited. They cannot penetrate the dual barriers of the stratum corneum and basement membrane under ultrasound to deliver drugs non-invasively to deep tissues.

[0009] Microbubbles (MBs) are widely used in clinical practice to enhance ultrasound imaging contrast and deliver drugs, genes, and gases to target tissues. Under ultrasound, the volume of microbubbles changes during expansion and compression, a process known as microbubble cavitation. With low-intensity ultrasound agitation, microbubbles can generate stable cavitation, enhancing cell and vascular permeability and effectively opening the double skin barrier. However, currently developed ultrasound microbubble coupling agents still suffer from shallow penetration depth, failing to reach deeper tissue layers.

[0010] Patent document CN 1943541 A provides an ultrasonic microbubble skin penetration enhancer, which is an ultrasonic microbubble that can burst under ultrasonic triggering. The ultrasonic microbubble can generate penetration enhancement and massage functions, enhance the skin's ability to penetrate active substances into the skin, and improve the skin's absorption function. However, the drug penetration depth that this ultrasonic microbubble skin penetration enhancer can achieve is only about 300 μm, which is still relatively low, and the penetration time is also relatively long.

[0011] Patent document CN 105435224 A discloses a microbubble coupling agent composed of a hydrophilic gelling agent, a microbubble film-forming agent, a surfactant, and a solvent. This microbubble coupling agent, while performing ultrasound coupling, can also promote the opening of the skin barrier, improve the efficiency of ultrasound drug penetration therapy, and realize the extradermal application of ultrasound microbubbles. However, the drug penetration depth of this method can only reach 300-350 μm, and the penetration depth and penetration efficiency still need to be improved.

[0012] On the other hand, existing ultrasonic coupling agents have poor adhesion and lack self-healing properties, failing to restore their original state well after use. Their performance in this aspect still needs to be improved.

[0013] Therefore, how to provide a new ultrasound microbubble coupling agent to achieve deeper skin penetration, improve the drug penetration efficiency of ultrasound non-invasive drug delivery, and enhance the adhesion and self-healing properties of the ultrasound coupling agent has become an urgent technical problem to be solved. Summary of the Invention

[0014] The present invention aims to solve the aforementioned technical problems by providing an ultrasonic nanobubble coupling agent, its preparation method, and its application. The technical objective of this invention is twofold: firstly, to improve the skin penetration depth of existing ultrasonic microbubble coupling agents and achieve rapid penetration, thereby increasing drug penetration efficiency; and secondly, to enhance the adhesion and self-healing properties of existing ultrasonic microbubble coupling agents.

[0015] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0016] This invention first provides a method for preparing an ultrasonic nanobubble coupling agent, which includes the following steps:

[0017] (1) Using aminated PLGA, celecoxib and dichloromethane as the oil phase and polyvinyl alcohol and perfluorohexane as the aqueous phase, celecoxib-loaded nanobubbles were obtained by two ultrasonic treatments using a double emulsion method, denoted as Cel@PFH@PLGA-NH2NBs;

[0018] (2) Hyaluronic acid was aldehyde-modified and amino-modified to prepare HA-CHO and HA-NH2. HA-CHO and HA-NH2 were mixed and gelled to prepare HA-CHO / HA-NH2 hydrogel.

[0019] (3) Mix Cel@PFH@PLGA-NH2 NBs with HA-CHO / HA-NH2 hydrogel solution and carry out Schiff base reaction to prepare ultrasonic celecoxib hyaluronic acid hydrogel, which is ultrasonic nanobubble coupling agent.

[0020] The preparation method provided by this invention uses Cel@PFH@PLGA-NH2 nanobubbles prepared by a double emulsification method and aminoated hyaluronic acid as raw materials. A novel ultrasonic nanobubble coupling agent is prepared through a Schiff base reaction between the nanobubbles and aldehyde-based hyaluronic acid hydrogels. As is well known, truly non-invasive drug delivery involves effectively and controllably delivering drugs into the body for circulation and absorption without disrupting the skin barrier, through the skin's dual barriers (the stratum corneum barrier and the basement membrane barrier between the epidermis and dermis). The ultrasonic nanobubble coupling agent of this invention amplifies the cavitation effect of ultrasound, allowing drugs to effectively cross the skin's dual barriers and be delivered to deeper layers. This significantly increases the depth of drug penetration and enables rapid penetration in a very short time, achieving truly non-invasive drug delivery.

[0021] In an in vitro porcine skin model, when the ultrasound parameters were set to 2 W, 650 kHz, 50% duty cycle, and 20 min, the ultrasound nanobubble coupling agent of this invention achieved an effective drug penetration depth of 728 μm, enabling efficient and non-invasive drug delivery to deeper layers. Comparative studies, however, show that existing ultrasound microbubble coupling agents can only achieve an effective drug penetration depth of 300-350 μm, and the penetration rate is relatively slow.

[0022] Furthermore, the weight ratio of the amino-modified PLGA to celecoxib in step (1) is 5:1.

[0023] Furthermore, the polyvinyl alcohol in step (1) has a mass-volume concentration of 1% in the aqueous phase, w / v.

[0024] Furthermore, the preparation method of HA-CHO in step (2) is as follows: hyaluronic acid and sodium periodate are dissolved in water, stirred at 25°C for 4 hours in the dark, the reaction is quenched with ethylene glycol, the solution is dialyzed, and the product is freeze-dried to obtain the product.

[0025] Furthermore, the preparation method of HA-NH2 in step (2) is as follows: hyaluronic acid is dissolved in water, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are dissolved in a mixed solution of dimethyl sulfoxide and water at a volume ratio of 1:1, and then mixed with the hyaluronic acid solution and reacted for 1 hour. Dihydrazine adipate is added, the pH is adjusted to 6.8, and dialyzed. The resulting solution is centrifuged, the supernatant is dialyzed at room temperature, and the resulting product is freeze-dried to obtain the final product.

[0026] Furthermore, the mass ratio of HA-NH2 and HA-CHO in step (2) is 2:5.

[0027] Furthermore, the volume ratio of Cel@PFH@PLGA-NH2 NBs to HA-CHO / HA-NH2 hydrogel in step (3) is 2:1.

[0028] Furthermore, the Schiff base reaction time in step (3) is 10 min.

[0029] A second objective of this invention is to provide a nanobubble ultrasonic coupling agent prepared by any of the methods described above.

[0030] A third objective of this invention is to provide an application of the nanobubble ultrasound coupling agent as described above, which is to use the nanobubble ultrasound coupling agent as a drug carrier for non-invasive drug delivery.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention is the first to use a dual emulsification method to prepare Cel@PFH@PLGA-NH2 nanobubbles, and then synthesizes HA-NH2 and HA-CHO through carboxyl-amino coupling reaction and oxidation reaction, respectively. Then, Cel@PFH@PLGA-NH2 nanobubbles and HA-NH2 are cross-linked with HA-CHO through Schiff base reaction and dynamic covalent cross-linking to construct an ultrasonic coupling agent based on nanobubbles. By amplifying the ultrasonic cavitation effect, the dual skin barrier is opened, and the drug is gradually pushed into the dermis to achieve deep non-invasive drug delivery. The effective penetration depth of the drug can reach 728 μm.

[0033] (2) This ultrasonic nanobubble coupling agent also has excellent adhesion, self-healing and injectability. This ultrasonic nanobubble coupling agent provides better application for non-invasive drug delivery platforms and disease treatment. Attached Figure Description

[0034] Figure 1The diagram illustrates the principle of effective, non-invasive, deep drug delivery using ultrasound nanobubble coupling agents; (A) Preparation process of celecoxib-loaded PLGA nanobubbles (Cel-PLGA NBs); (B) Synthesis process of UCH hydrogel; (C) Multifunctional application examples of ultrasound nanobubble coupling agents (ultrasound imaging, ultrasound examination, non-invasive drug delivery).

[0035] Figure 2 Nuclear magnetic resonance of HA-NH2 ( 1 (H-NMR) hydrogen spectrum.

[0036] Figure 3 The infrared spectrum of HA-NH2.

[0037] Figure 4 Characterization of ultrasonic nanobubble coupling agents; (A) Characterization of ultrasonic nanobubble coupling agents; (B, C) Digital camera photographs of application examples of nanobubbles and commercial coupling agents; (D) Scanning electron microscope images of Cel-PLGA NBs; (E) Scanning electron microscope images of ultrasonic nanobubble coupling agents; (F, G) Phase transition process of Cel-PLGA NBs; (H) Fourier transform infrared spectroscopy analysis of different samples; (I) Particle size distribution diagram of Cel-PFH-PLGA NBs; (J, K) Rheological analysis of ultrasonic nanobubble coupling agents; (L) Schematic diagram of the adhesion ability of nanobubble coupling agents and commercial coupling agents; (M, N) Application examples of the adhesion ability of nanobubbles and commercial coupling agents on pigskin.

[0038] Figure 5 Transmission electron microscopy images of Cel-PLGA NBs.

[0039] Figure 6 Dynamic light scattering analysis of PLGA nanobubbles.

[0040] Figure 7 and Figure 8 Rheological tests for commercial coupling agents.

[0041] Figure 9 Rheological testing of ultrasonic nanobubble coupling agents, including shear and cyclic strain.

[0042] Figure 10 (A, B) Chitosan in vitro model of ultrasonic nanobubble coupling agent for effective non-invasive deep drug delivery; (C) Schematic diagram of ultrasonic nanobubble coupling agent release experiment; (D, E) Release curve of ultrasonic nanobubble coupling agent; (F) Schematic diagram of ultrasonic nanobubble coupling agent experiment; (G) Representative image of ultrasonic nanobubble coupling agent in nude mouse in vivo imaging system (IVIS).

[0043] Figure 11(A) Schematic diagram of an experiment on non-invasive drug delivery using ultrasonic nanobubble coupling agents; (B) Immunofluorescence staining image of an in vitro porcine skin model using ultrasonic nanobubble coupling agents; (C) Mathematical simulation image of ultrasonic nanobubble coupling agents.

[0044] Figure 12 This study investigates the ultrasonic thermal effect using pig skin as an in vitro experimental model.

[0045] Figure 13 Representative images of ultrasonic imaging of nanobubble coupling agents; (A, D) Schematic diagram of ultrasonic imaging experiment of nanobubble coupling agents; (B, C) Representative images of ultrasonic nanobubble coupling agents and commercial coupling agents in different tissues; (E, F) Representative images of ultrasonic nanobubble coupling agents and commercial coupling agents after rotation in different tissues; (GJ) Representative images of ultrasonic nanobubble coupling agents and commercial coupling agents after torsional changes in body position in different tissues.

[0046] Figure 14 To assess the biocompatibility of the ultrasonic nanobubble coupling agent and its role in relieving Achilles tendon adhesions; (A) Live / dead staining of different groups of F208 fibroblasts on days 1, 3, and 5; (B) Cytoskeleton staining of different groups of F208 fibroblasts on days 1, 3, and 5; (C, D) Immunofluorescence staining results of F208 fibroblasts: red (cytoskeleton of F208 fibroblasts), green (Cox-2, Col-1 markers), blue (nucleus); (E) Cell viability (CCK-8) of F208 fibroblasts on days 1 and 4 (NS, no significant difference); (F) Cytotoxicity of F208 fibroblasts at 24 h; (G, h) Fluorescence intensity was compared using one-way ANOVA and Tukey post-hoc test for multiple comparisons; (Data are expressed as mean ± standard deviation, and were analyzed using one-way ANOVA or two-way ANOVA, NS, no significant difference, ****, p < 0.0001).

[0047] Figure 15 To assess the in vivo performance of ultrasound nanobubble coupling agents in preventing tendon adhesions; (A) Rat tendon adhesion model and surgical schematic diagram; (B, C, D) Macroscopic and ultrasound images of Achilles tendon adhesions in rats at 7, 14, and 28 days after treatment; (E, F) Macroscopic and ultrasound scores of Achilles tendon adhesions in different treatment groups at 7, 14, and 28 days after treatment; Achilles tendon adhesions in rats were graded from 0 to 5, and the width of the Achilles tendon was mainly statistically analyzed in ultrasound images; (**p<0.01, ***p<0.001, ****p<0.0001; data are expressed as mean ± SD, n = 6; NS, no significant difference).

[0048] Figure 16For in vivo histological analysis; (A, B) Representative images of H&E staining and Masson staining at 1W and 2W; (C, D) Immunofluorescence staining of rat Achilles tendon tissue, representative images of COX-2 and Col-III; quantitative analysis of expression differences among related proteins; (EH) Histological score and quantitative immunofluorescence analysis of rat Achilles tendon tissue; (***p<0.001, ****p<0.0001; data are expressed as mean ± SD, n = 6; NS, no significant difference).

[0049] Figure 17 Images of H&E-stained and Masson-stained tissue sections after 28 days.

[0050] Figure 18 For in vitro immunofluorescence detection of Col-3.

[0051] Figure 19 Immunofluorescence staining of in vivo tissues with Col-1 as the marker.

[0052] Figure 20 This is an in vivo immunofluorescence image of a 28-day-old tissue section targeting the Col-III protein. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0054] Example 1

[0055] I. Experimental Materials and Methods

[0056] 1. Preparation and characterization of celecoxib-loaded nanobubbles (Cel@PFH@PLGA-NH2NBs)

[0057] PLGA-NH2 nanobubbles (NBs) loaded with celecoxib (Cel) were synthesized using a two-emulsion method. The oil phase consisted of 50 mg PLGA-NH2 (Sunlipo Nano Tech, China), 10 mg Cel, and 2 ml dichloromethane (Aladdin, Shanghai, China). 1% (w / v) polyvinyl alcohol (PVA) and perfluorohexane (PFH) were added to the mixture, followed by two ultrasonic treatments. The resulting suspension was treated with 10 mL of 2% (v / v) isopropanol solution and continuously stirred at 25 °C for 4 hours. The dichloromethane was then evaporated to obtain the celecoxib-loaded nanobubbles (denoted as Cel@PFH@PLGA-NH2NBs).

[0058] The morphology, composition, and particle size of nanobubbles were investigated using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and dynamic light scattering (DLS) to comprehensively describe the formation process and its effects. The stability and phase transition of Cel@PFH@PLGA-NH2NBs were detected using photoacoustic analysis. The effect of ultrasonic irradiation on Cel@PFH@PLGA-NH2NBs was also examined using photoacoustic analysis. After ultrasonic irradiation, 50 mg / mL of water or UGV hydrogel was dissolved in 3% (w / v) agarose gel. The encapsulation efficiency was measured according to the method described in the reference “Xiaoyu Han, Shuyu Chen, Zhengwei Cai, Ying Zhu, Weiwei Yi, Mengtong Guan, Bo Liao, Ying Zhang, Jieliang Shen, Wenguo Cui, Dingqun Bai. Adv. Funct. Mater. 2023,33, 2213008”.

[0059] 2. Preparation of HA-CHO / HA-NH2 and UCH hydrogels

[0060] To generate HA-CHO, 1 g of hyaluronic acid (HA, Macklin, Shanghai, China) and 450 mg of sodium periodate (Macklin, Shanghai, China) were dissolved in 90 mL of ddH2O. The solution was stirred at 25°C in the dark for 4 hours, and then quenched with 500 μL of ethylene glycol (Aladdin, Shanghai, China). The solution was then dialyzed against ddH2O for 3 days, with the medium changed 3 times a day, and then freeze-dried and stored at -20°C.

[0061] To synthesize HA-NH2, 2 g of HA (Macklin, Shanghai, China) was completely dissolved in 100 ml of ddH2O. 4.58 g of HOBT (1-hydroxybenzotriazole, Macklin, Shanghai, China) and 2.02 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, Macklin, Shanghai, China) were dissolved in a 1:1 volume ratio of DMSO (dimethyl sulfoxide) and ddH2O, and this solution was added dropwise to the HA solution to activate the carboxyl groups. After 1 hour, 4.58 g of dihydrazine adipic acid (Macklin, Shanghai, China) was added to adjust the pH to 6.8. After dialyzing with ddH2O for 3 days, the resulting solution was centrifuged at 7000 rpm for 15 minutes. The supernatant was then transferred to a dialysis bag with a molecular weight of 3500 kDa and dialyzed at room temperature for 3 days, changing the medium three times a day. After lyophilization, the solution was stored at -20°C.

[0062] Finally, 1.6 mg of Cel@PFH@PLGA-NH2 nanobubbles were loaded into HA-NH2 (2%, m / v) / HA-CHO (5%, m / v) (ratio 2:1, v / v) hydrogel and Schiff base reaction was carried out, which was completed in about 10 min to obtain UCH hydrogel (ultrasonic celecoxib hyaluronic acid hydrogel).

[0063] 3. Characterization of UCH hydrogel and non-invasive drug permeation experiment

[0064] The surface morphology, chemical groups, injectability, and cyclic strain of the UCH hydrogel were investigated using scanning electron microscopy, a digital camera, infrared analysis, and rheology. Furthermore, the adhesion of the UCH hydrogel to a commercial coupling agent (Chengxin Medical Ultrasonic Coupling Agent TM-100, manufactured by Jinnankai Food and Drug Administration) was studied through in vitro hydrogel adhesion experiments after rotation and torsion of ex vivo porcine skin. Ultrasound has a significant thermal effect; thermal imaging technology was used to measure the heat generated by different ultrasonic intensities in vitro to determine safe and reliable ultrasonic parameters. The ultrasonic parameters were 650 kHz, 50% duty cycle, and ultrasonic power of 0 W, 1 W, 2 W, 3 W, and 4 W.

[0065] The non-invasive drug penetration depth of ultrasound into UCH hydrogel was verified through in vivo chitosan gel experiments, release experiments, porcine skin experiments, mathematical modeling simulations, and BALB / c nude in vivo imaging experiments. UCH hydrogel was placed on a chitosan (0.5%, m / v) solution, and cavitation was performed on the UCH hydrogel using a low-intensity focused ultrasound (LIFU, model UTG 1025, Institute of Ultrasound Imaging, Chongqing Academy of Medical Sciences, China), with the intensity gradually increased. Ultrasonic cavitation was performed on the UCH hydrogel using the ultrasonic cavitation method, with the ultrasonic intensity gradually increasing. The ultrasonic parameters were 650 kHz, 50% duty cycle, and ultrasonic power of 0 W, 1 W, 2 W, 3 W, and 4 W.

[0066] Secondly, a transdermal drug delivery model was used. SD rat skin was covered with 25 ml PBS centrifuge tubes, and UCH hydrogel was applied to the entire skin layer using LIFU (650 kHz, 50% duty cycle, 2 W) to stimulate drug release. The process was repeated for 2 weeks, with samples collected every minute and 1 ml of fresh PBS added to the reservoir to maintain a constant volume. Celecoxib concentrations were determined using a 249 nm UV spectrophotometer, and corresponding release curves were plotted. Similarly, in vivo animal imaging was used to investigate the non-invasive drug penetration efficiency of the UCH hydrogel. BALB / c nude mice (male, 6 weeks old) were obtained from the Animal Center of Shanghai Jiao Tong University School of Medicine, and the experimental protocol was approved by the Animal Health Committee of Shanghai Jiao Tong University School of Medicine. All nude mice and rats were housed in a specific pathogen-free (SPF) environment. Rats were randomly divided into five groups and sonicated with indocyanine green (ICG)-labeled UCH hydrogel at 650 kHz, 50% duty cycle, and 2 W for 0, 5, 10, 15, and 20 minutes using an in vivo imaging system (IVIS). Ultrasonic non-invasive drug delivery was conducted using excised porcine skin to evaluate the drug delivery depth of the UCH hydrogel. All porcine skins were purchased from a Shanghai fresh food market. The sonication parameters were 650 kHz, 50% duty cycle, and 2 W, with sonication times of 0, 5, 10, 15, and 20 minutes. After sonication, all tissue specimens were washed, frozen completely with dry ice or liquid nitrogen, sectioned, and immunofluorescence stained to detect drug penetration depth. Mathematical modeling was used to simulate the experimental results using excised porcine skin.

[0067] 4. External ultrasound imaging

[0068] Thirty SD rats (12 weeks old, 250g) and 30 male BALB / c nude mice (6 weeks old) were obtained from the Animal Center of Shanghai Jiao Tong University School of Medicine. The experimental protocol was approved by the Animal Health Committee of Shanghai Jiao Tong University School of Medicine. All animals were anesthetized with isoflurane, and imaging was performed using UCH hydrogel or commercially available coupling agents to evaluate their imaging effects on the Achilles tendon, gastrocnemius muscle, stomach, and kidneys of the target animals.

[0069] 5. Cell Culture

[0070] F208 fibroblasts were provided by Shanghai Jiao Tong University. Cells were cultured at 37°C in α-basal medium (α-MEM, GIBCO) supplemented with 10% fetal bovine serum under a 5% CO2 atmosphere. The α-MEM basal medium was changed every 2 days.

[0071] 6. Biocompatibility of UCH hydrogel and its effect on relieving Achilles tendon adhesions

[0072] The proliferation and toxicity of UCH hydrogel on F208 fibroblasts were comprehensively evaluated and studied using live / dead cell labeling, cytoskeleton staining, CCK-8 proliferation assay, and lactate dehydrogenase (LDH) toxicity assay. All experiments were performed using rat full-thickness skin barrier extract. First, cell viability was measured at 1, 3, and 5 days using a live / dead cell labeling kit (Invitrogen, L3224, USA) containing the fluorescent dyes Calcein-AM (green) and propidium iodide (PI) (red). Fluorescence images were captured using a confocal microscope (Leica).

[0073] To assess the effect of sample-induced cytotoxicity on cell proliferation, lactate dehydrogenase (LDH, Beyotime, China) and cell counting kit-8 (CCK-8, Dongjingdu, Japan) were used. Cytoskeleton staining (Yeasen, China) was used to evaluate the adhesion and morphology of F208 fibroblasts on the samples. After 1, 3, and 5 days of culture, cells were infiltrated with 0.1% (v / v) Triton X-100, fixed with 4% polyformaldehyde, and stained with 0.2 mL DAPI and 5 g / mL Alexa Fluor 594phalloidin, respectively, to identify actin and the nucleus. The cellular actin cytoskeleton and nucleus were observed using a fluorescence microscope (Leica, Japan).

[0074] To detect the in vivo anti-adhesion effect of UCH hydrogel on the Achilles tendon, immunofluorescence was primarily used. In short, F208 fibroblasts at 5 × 10⁸ cells / year... 4 Cell / well density culture. After culturing in the extraction buffer for 48 hours, the samples were stained with immunofluorescence. Cells were first fixed with 4% paraformaldehyde, infiltrated with 0.1% (v / v) Triton X-100 for 15 minutes, blocked with 1% BCA for 30 minutes, and then incubated with primary antibody and fluorescent secondary antibody. The protein expression of Col I (ab34710, Abcam), Cox-2 (proteintech 12375-1-AP), and Col III (ab184993, Abcam) was then observed using confocal microscopy.

[0075] 7. Rat Achilles tendon adhesion model

[0076] All animal experiments were approved by Shanghai Jiao Tong University School of Medicine. SD rats weighing 250-300 g were selected as experimental subjects and randomly divided into four groups: control group, celecoxib hyaluronic acid hydrogel (CH hydrogel) group, ultrasonic celecoxib PLGA nanobubbles (UCP NBs) group, and UCH hydrogel group. A total of 80 SD rats were used in the experiment.

[0077] The establishment of the rat Achilles tendon adhesion model is briefly described as follows: The rats were anesthetized intraperitoneally with 0.3% sodium pentobarbital (0.5 mL / 100g). Leg hair was shaved, and the area was disinfected with povidone-iodine. The skin above the Achilles tendon was also disinfected with a scalpel. A 15 mm longitudinal incision was made to expose the Achilles tendon, and a 5 mm lesion was made along the tendon direction. The skin was sutured and disinfected with povidone-iodine. Penicillin at a dose of 20 mg / kg was injected to prevent infection. Rats were sacrificed 7, 14, and 28 days after implantation. The degree of Achilles tendon adhesion was observed visually and by ultrasound. The Achilles tendon was then removed, fixed with 4% paraformaldehyde, sectioned, embedded, and subjected to histological analysis. The Achilles tendon adhesion score is shown in Tables 1 and 2 below.

[0078] Table 1 Tendon adhesion scoring scheme

[0079]

[0080] Table 2 Histological Adhesion Scoring Scheme

[0081]

[0082] 8. H&E staining, Massen staining, immunofluorescence staining

[0083] Tissue samples were fixed with 4% paraformaldehyde, dehydrated with ethanol, and embedded in paraffin to a thickness of 4 µm. Histopathological features were detected by H&E staining and Masson staining. Immunofluorescence staining was performed, followed by dewaxing, rehydration, and washing of tissue sections, then blocking with goat serum. Next, tissue sections were incubated overnight with primary antibodies, followed by Alexa fluorescence-conjugated secondary antibody (for immunofluorescence staining). Finally, tissue sections were observed under a fluorescence microscope. The following primary antibodies were used: Immunohistochemical image scoring was performed using Image J software for Col-1 (ab34710, Abcam), COX-2 (proteintech 12375-1-AP), and COL-III (ab184993, Abcam).

[0084] 9. Data Statistics

[0085] Experimental results are presented as the mean standard deviation after at least three replicates for each segment. Statistical analysis was performed using Origin software and GraphPadPrism 8.0. Student's t-test was used to compare the results between the two groups. One-way or two-way ANOVA was used to compare the results between different groups. A p-value less than 0.05 was considered statistically significant.

[0086] II. Experimental Results

[0087] 1. Characterization and ultrasonic responsiveness of nanobubble ultrasonic coupling agents

[0088] The principle diagram of the effective, non-invasive, deep drug delivery of the ultrasonic nanobubble coupling agent provided by this invention is shown below. Figure 1 .

[0089] As a topical patch, the nanobubble ultrasonic coupling agent requires good adhesion, lubricity, injectability, self-healing properties, acoustic wave conduction, and biocompatibility. This invention selects hyaluronic acid (HA) as the substrate for the coupling agent patch, which possesses good biocompatibility and low immunogenicity, and is an important component of natural ECM. By modifying hyaluronic acid with aldehyde and amino groups, the nanobubble ultrasonic coupling agent acquires injectability and good adhesion. To verify the successful modification of HA with aldehyde and amino groups, proton nuclear magnetic resonance (PNMR) was performed. 1 1H-NMR spectral analysis. Figure 2 Three new peaks appeared in the spectrum of HA-NH2, which were provided by the NHNHCO-CH2- (2H, 2.0-2.4 ppm) of hydrazine in HA-NH2. Furthermore, infrared spectroscopy showed that HA-NH2 exhibited peaks at 3400 cm⁻¹. -1 There is a broadened peak nearby, HA-CHO at 1740 cm. -1 There is an additional peak nearby ( Figure 3 These results confirm the successful modification of the hydrogel precursors HA-NH2 and HA-CHO. In subsequent gel formation experiments, the substrate material for the ultrasonic nanobubble coupling agent was successfully constructed using the Schiff base reaction through the above modification.

[0090] To enhance the ultrasonic cavitation effect of the ultrasonic nanobubble coupling agent, the ultrasonic cavitation effect was amplified to effectively penetrate the skin's dual barriers and achieve deep, non-invasive drug delivery. Celecoxib-loaded PLGA nanobubbles (Cel-PFH-PLGA NBs) were prepared using a dual emulsification technique. The surface morphology and particle size distribution of the synthesized samples were observed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and DLS analysis. TEM and SEM data showed that the Cel-PFH-PLGA NBs were uniform in size and stably distributed throughout the field of view. Figure 4 Part D, Figure 5 The average diameter of Cel-PFH-PLGA NBs, measured using DLS, was 254.5 nm, with a polydispersity index (PDI) of 0.190 and a surface potential of -8.07 mV. Figure 4 It is worth noting that the PFH-PLGA NBs have a particle size of 192.4 nm, a PDI of 0.190, and a surface potential of -23.8 mV. Figure 6 These results demonstrate that, compared to PFH-PLGA NBs, Cel was successfully loaded onto the PLGA shell surface, and the surface potential and particle size of the nanoparticles were altered to some extent.

[0091] Furthermore, FTIR analysis of Cel-PFH-PLGA NBs revealed that the characteristic peak of Cel-PFH-PLGA NBs is located at 3250 cm⁻¹. -1 Around 1000 cm⁻¹, the characteristic peak of PLGA NBs is located at 1000 cm⁻¹. -1 and 3000 cm -1 The left and right sides also prove that Cel was successfully loaded into Cel-PFH-PLGA NBs. Figure 4 (H portion). Similarly, the cell encapsulation efficiency was determined by ultraviolet spectrophotometry at 85.9%. To detect the ultrasonic responsiveness of Cel-PFH-PLGA NBs, we directly applied ultrasound in vitro. We observed that as the ultrasonic intensity increased, the diameter of the nanobubbles gradually increased, even reaching the micrometer scale. Figure 4 (Part F in the middle). The phase transition (PT) effect of nanobubbles was detected in B-MODE and Contrast modes, respectively. Figure 4 As shown in section G, more nanobubbles are vaporized with increasing ultrasonic intensity, which is consistent with the results under light microscopy, indicating that the nanobubble ultrasonic coupling agent has good ultrasonic responsiveness.

[0092] Finally, using aldehyde-based hyaluronic acid hydrogel as a raw material, the Cel-PFH-PLGA NBs nanobubbles prepared by the double emulsification method were chemically cross-linked with amino hyaluronic acid via the Schiff base reaction and dynamic covalent bonding. To verify the success of the grafting, scanning electron microscopy analysis was performed on the samples. The results showed that, compared with the blank HA hydrogel, Cel-PFH-PLGA NBs not only exhibited a uniform porous structure, but also showed that Cel-PFH-PLGA NBs adhered to the composite hydrogel patch, thus verifying the successful loading of Cel-PFH-PLGA NBs into the ultrasonic nanobubble coupling agent. Figure 4 Part E of the middle section).

[0093] The injection, lubrication, and adhesion properties of an ultrasonic nanobubble coupling agent were studied. First, the coupling agent was encapsulated. Results showed that the ultrasonic nanobubble coupling agent was successfully extruded from the encapsulation bottle, exhibiting no difference from commercial coupling agents and demonstrating good injectability. Figure 4(AC section). Subsequent images show that the nanobubble ultrasound coupling agent exhibits excellent lubrication properties and acts as a bridge for sound wave transmission between the ultrasound probe and the skin. Notably, the hydrogen and dynamic Schiff base bonds between the ultrasound nanobubble coupling agent and the skin result in excellent adhesion. Adhesion experiments on pigskin also demonstrated that the ultrasound nanobubble coupling agent exhibits stronger adhesion than commercially available coupling agents. Even after immersion in ultrapure water and pigskin deformation experiments, it remained firmly adhered to the pigskin, making it a superior coupling agent. Figure 4 (Middle LN part).

[0094] Finally, rheological tests were performed on the ultrasonic nanobubble coupling agent. It was found that under certain mechanical pressure, the coupling agent patch disrupted the double bonds generated by the Schiff base reaction, resulting in a low hydrogel modulus. Compared with the previous gel modulus, it exhibited good self-healing properties, which is beneficial to the conduction of ultrasonic cavitation effect. Figure 4 JK section Figure 7 and Figure 8 In rheological tests, the ultrasonic nanobubble coupling agent was more stable than commercially available coupling agents.

[0095] In summary, ultrasonic nanobubble coupling agents can effectively fulfill their coupling agent function, significantly improving their performance in terms of adhesion, injectability, and self-healing properties. Furthermore, in the presence of amination-modified nanobubbles, the mechanical properties of the coupling agent increase from 30 Pa to 45 Pa with increasing nanobubble count. This may be because the increased loading of amination-modified nanobubbles enhances the binding sites for the Schiff base reaction. Figure 9 Coupling agents improve the mechanical properties of materials to a certain extent, and can still play their role in transmitting ultrasonic energy without affecting their self-healing properties.

[0096] Nanobubble ultrasound coupling agents effectively penetrate the skin's dual barriers by amplifying the ultrasonic cavitation effect.

[0097] Under ultrasound, the volume of microbubbles changes during expansion and compression, a process known as microbubble cavitation. Notably, under low-intensity ultrasound stimulation, microbubbles can generate stable cavitation, thereby enhancing cell and blood vessel permeability. Therefore, we propose using ultrasound-guided nanobubble coupling agents to open the pore channels of the stratum corneum and basement membrane of the skin under ultrasound.

[0098] To verify the transdermal drug delivery efficiency of the ultrasonic nanobubble coupling agent, various methods were employed, including in vivo chitosan gel simulation experiments, release tests, in vivo animal imaging, in vitro porcine skin models, and digital modeling, to validate the transdermal drug delivery efficiency of the ultrasonic nanobubble coupling agent from multiple perspectives. Firstly, in a 0.5% chitosan in vitro solution model, it was observed that the penetration of the dye-loaded coupling agent patch gradually increased with increasing ultrasonic intensity. Figure 10 (Parts A and B). Furthermore, under the action of an ultrasonic switch, the dye in the ultrasonic nanobubble coupling agent is significantly induced to penetrate into the chitosan model through ultrasonic cavitation. Moreover, as the ultrasonic intensity increases, the dye penetrates deeper into the chitosan model, indicating that the ultrasonic nanobubble coupling agent possesses good ultrasonic responsiveness. It is noteworthy that the dye in the ultrasonic nanobubble coupling agent, under the action of an ultrasonic switch, is significantly induced to penetrate into the chitosan model through ultrasonic cavitation. Furthermore, as the ultrasonic intensity increases, the dye penetrates deeper into the chitosan model, further demonstrating that the ultrasonic nanobubble coupling agent exhibits good ultrasonic responsiveness.

[0099] Secondly, the transdermal drug delivery efficiency of the ultrasonic nanobubble coupling agent was tested through release experiments. Figure 10 (CE section). First, the entire rat skin was sealed in a centrifuge tube filled with PBS buffer. Then, the coupling agents from different groups were evenly applied to the rat skin and subjected to ultrasound treatment. The experimental results showed that no drug penetration was detected in the coupling agent patch without ultrasound treatment under rat skin blockage. Under ultrasound exposure, the drug release of both the commercial coupling agent and the ultrasound nanobubble coupling agent was significant. However, it is noteworthy that, at the same ultrasound intensity, the transdermal permeability of the ultrasound nanobubble coupling agent was 2.17 times that of HA hydrogel alone. This indicates that the nanobubbles further amplified the cavitation effect of ultrasound after ultrasound treatment, improving drug delivery efficiency.

[0100] Interestingly, in Figure 10 As seen in Part D, no drug penetration was detected during the time the ultrasound was stopped, concluding that ultrasound is a "key factor" for drug penetration. In in vivo imaging experiments on nude mice, it was observed that at a certain ultrasound intensity, the dye fluorescence intensity increased with increasing exposure time, which also demonstrates the transdermal drug penetration effect of the ultrasound nanobubble coupling agent. Figure 10 (Parts F and G).

[0101] Furthermore, to verify the drug penetration effect of the ultrasonic nanobubble coupling agent in various aspects, an in vitro drug penetration test was conducted using excised pig skin. Pig skin is similar to human skin in structure and chemical composition, and can well simulate the reaction of human skin. In addition, the drug penetration depth was quantified using biological methods such as sectioning and immunofluorescence staining. The results showed that red indicates the presence of the drug dye, and the drug penetration trend remained the same over time. Compared with commercially available ultrasonic nanobubble coupling agents, the drug penetration depth increased by 3.1 times. Compared with the 234 μm of commercial coupling agents, the coupling agent of this invention achieved a penetration depth of 728 μm under ultrasound for 20 minutes. Figure 11 Part A). To better track the drug penetration trajectory, a mathematical model was used to simulate the transdermal penetration effect of the drug under various conditions. The results were consistent with those obtained from isolated pig skin, which more intuitively illustrates that the nanobubble ultrasonic coupling agent synthesized in this invention greatly improves the transdermal penetration efficiency of the drug through the cavitation effect. Figure 11 Part B of the middle section.

[0102] Study on the biocompatibility of ultrasonic nanobubble coupling agents

[0103] To investigate the biocompatibility of ultrasonic nanobubble coupling agents, in vitro thermal imaging, in vitro development, and biocompatibility tests were performed on hydrogel coupling agent patches. It is well known that ultrasound generates a large amount of heat during contact, which can cause thermal damage to the skin. Studies have shown that temperatures below 43°C do not damage the skin or cells. Therefore, thermal imaging was used to conduct experiments on isolated pig skin to study the temperature generation under different ultrasound intensities. The results are as follows: Figure 12 As shown in the figure, the critical damage temperature reached 43℃ after 1 minute of exposure at 3W power, and 33.8℃ after 20 minutes of exposure at 2W power. Therefore, 2W of ultrasonic intensity was chosen for subsequent experiments while ensuring drug penetration efficiency.

[0104] To investigate the ultrasonic imaging effect of ultrasonic nanobubble coupling agents, this study explored whether novel coupling agents could improve the transmission efficiency between ultrasound waves and human tissue, thereby enhancing the ability of sound waves to penetrate deep into human tissue and better observe and diagnose internal structures. Figure 13 Part A). In vitro multimodal ultrasound examination of internal organs in rats and nude mice was used to study and compare the sound transmission rate of the ultrasound nanobubble coupling agent. Figure 13 As shown in section B, the fibrous arrangement of the Achilles tendon tissue is clearly visible, and its echo intensity is significantly different from that of the surrounding tissue, indicating that the ultrasonic nanobubble coupling agent successfully transferred the acoustic energy. Figure 13As shown in section C, the ultrasound nanobubble coupling agent and the commercially available coupling agent have a more significant impact on the morphology of the gastrocnemius muscle, clearly displaying its tissue contours. Notably, the diaphragm of the gastrocnemius muscle is also clearly visible. Interestingly, in ultrasound images of the stomach ( Figure 13 In section D, the fluid level of the gastric contents can be clearly observed, reflecting that ultrasound nanobubble coupling agents can improve the speed and uniformity of ultrasound transmission in tissues, thereby achieving the required ultrasound imaging clarity. Figure 13 The corresponding phenomenon can also be observed in the ultrasound images of the renal parenchyma in the middle E region.

[0105] It is worth noting that, compared with traditional coupling agents, ultrasound nanobubble coupling agents exhibit superior adhesion. By changing the patient's position and the angle of the ultrasound probe, they remain in the target visualization position, avoiding the drawbacks of repeated application of conventional couplers. Furthermore, conventional coupling agents require repeated application to ensure the clarity of the ultrasound image. Results are as follows... Figure 13 As shown in section FJ, without the addition of new coupling agents, UCH hydrogel can still clearly display tissue contours and structural features even when the two coupling agents undergo positional changes such as torsion. In contrast, traditional coupling agents, due to insufficient adhesion and detachment, result in poor imaging; while they can generally display the contours of the target tissue, they cannot provide sufficient structural detail, significantly affecting the operator's interpretation of the ultrasound images.

[0106] Biocompatibility of Ultrasonic Nanobubble Coupling Agents

[0107] As a medical biomaterial, ultrasonic nanobubble coupling agents need to possess good biocompatibility. Improper use of ultrasonic coupling agents may have certain adverse effects on the human body, such as allergies and skin irritation. Fibroblasts are the main constituent cells of skin tissue; therefore, the biocompatibility of ultrasonic nanobubble coupling agents was assessed by detecting the live / dead staining of fibroblasts (F208), bone staining, CCK-8 levels, and lactate dehydrogenase (LDH).

[0108] The samples were divided into four groups: control group, CH hydrogel (Cel NBs@HA hydrogel) group, UCP NBs (Ultrasound@Cel NBs) group, and UCH hydrogel (Ultrasound@Cel NBs@HA hydrogel) group. The biocompatibility of the ultrasonic nanobubble coupling agents was investigated using an immersion culture method in this part of the experiment.

[0109] First, F208 fibroblasts were stained for both dead and live cells on days 1, 3, and 5 to observe cell state. Although some dead cells were observed, it was clear that all four groups of F208 fibroblasts exhibited good cell morphology and viability, and the differences in proliferation rate were not statistically significant. Figure 14 (Part A). Secondly, on days 1, 3, and 5, the cells cultured in the infusion solution were stained with cytoskeleton to examine the proliferation rate and morphology of F208 cells. This is the basis and prerequisite for verifying that the ultrasound nanobubble coupling agent is non-toxic and harmless. Cells stained on day 1 showed better extensibility and were round and spherical compared to cells stained on days 3 and 5. Clear cell morphology was visible after 3 days of culture, and cells completely covered the field of view after 5 days. There were no significant differences between the groups. Figure 14 Part B). Third, using the Cell Counting Kit-8 (CCK-8), the proliferation rate of F208 fibroblasts was normal among all groups, with no statistically significant difference between day 1 and day 4. The LDH study showed ( Figure 14 (Parts E and F) The ultrasonic nanobubble coupling agent is also a non-toxic biomaterial that meets the above standards. In summary, all F208 fibroblasts can maintain normal proliferative activity in the ultrasonic nanobubble coupling agent, which is a prerequisite for it to exert its coupling agent effect.

[0110] Reduce Achilles tendon adhesions by utilizing the cavitation effect of ultrasonic nanobubble coupling agents.

[0111] As is well known, Achilles tendon adhesions refer to the fibrous connections between the Achilles tendon and the bone, which usually occur after certain diseases or injuries. They increase tension between the Achilles tendon and the bone, leading to pain and impaired joint movement. These adhesions are typically caused by inflammation or trauma to the cells or tissues surrounding the Achilles tendon, resulting in the ligaments, fascia, or muscle tissue around the tendon sticking together, thus restricting the tendon's movement.

[0112] To investigate the effect of ultrasonic nanobubble coupling agents on efficient and non-invasive drug penetration through cavitation, a rat Achilles tendon adhesion model was used for validation. Celecoxib, a hydrophobic drug, is the first clinically approved COX-2-specific inhibitor, which may act by inhibiting cell proliferation. Since celecoxib itself does not easily cross two skin barriers, this invention utilizes ultrasonic nanobubble coupling agents for non-invasive drug delivery, reducing Achilles tendon adhesion in rats. The groups were: Control group, CH Hydrogel group, UCP NBs group, and UCH Hydrogel group.

[0113] First, a uniform 5 mm longitudinal incision was made on the rat's Achilles tendon. After irrigation, the incision was sutured and fixed with an elastic bandage. Transdermal ultrasound therapy was administered daily for 14 days, after which the external fixator was removed, thus establishing the Achilles tendon adhesion model. Rats were euthanized on days 7, 14, and 28, and Achilles tendon tissue was harvested, fixed, and observed using ultrasound. Figure 15 Part A of the middle section.

[0114] The results are as follows Figure 15 As shown in the BE section, Achilles tendon adhesion was first analyzed in each group using an Achilles tendon adhesion scoring system. Seven days post-operation, both the blank control and CH hydrogel groups showed extensive Achilles tendon adhesion, with no gaps between the Achilles tendon and the attached muscle tissue, indicating tight adhesion. Furthermore, palpation revealed hard lumps or nodules around the Achilles tendon, and ultrasound showed a widened and enlarged Achilles tendon outline with blurred muscle fibers. In contrast, while the UCP NBs and UCH hydrogel groups showed some adhesion due to injury, their adhesion scores were significantly lower than the control and CH hydrogel groups. On day 14 after modeling, the condition in the control and CH hydrogel groups showed no improvement and even worsened, with purplish-red markings and increased width visible on the Achilles tendon surface. In the UCP NBs and UCH hydrogel groups, adhesion was further reduced compared to day 7, reflecting successful local delivery of celecoxib and significantly reducing the degree of adhesion after Achilles tendon injury. Similarly, ultrasound imaging showed clear Achilles tendon fibers and appropriate width in the UCP NBs and UCH hydrogel groups. On day 28 of molding, the adhesions were somewhat relieved, possibly due to the removal of the external fixator.

[0115] To further evaluate the effect of ultrasonic nanobubble coupling agents on reducing Achilles tendon adhesions in rats, histological staining of the Achilles tendon tissue was performed. Figure 16 Parts A and B Figure 17 As shown, on postoperative day 7, HE staining revealed extensive adhesions between the tendon margin and surrounding granulation tissue in the control and CH hydrogel groups, indicating the occurrence of adhesions. On postoperative day 14, the fiber bundles became denser, and high-density fibrous adhesions further formed between the tendon and surrounding tissues. In contrast, the tendons treated with UCP NBs and UCH hydrogel showed only slight adhesions, with a clear gap between the tendon and skin. On postoperative day 28, adhesions in all groups were relieved, consistent with gross observations. Masson staining showed that on postoperative days 7 and 14, the control and CH hydrogel groups exhibited abundant blue-stained collagen hyperplasia, with collagen randomly distributed in the gap between the tendon and skin. In contrast, the UCP NBs and UCH hydrogel groups, especially on day 14, showed a clear gap between the tendon and skin, indicating that celecoxib was successfully delivered non-invasively and deeply via ultrasonic nanobubble coupling agent, significantly reducing Achilles tendon adhesions. Consistent with HE staining, the observations on postoperative day 28 were similar.

[0116] like Figure 16As shown in section CCD, the in vitro COX-2 immunofluorescence staining effect of celecoxib on rat Achilles tendon tissue and fibroblasts was evaluated. Under the influence of celecoxib, the COX-2 content expressed in fibroblasts decreased, and the in vivo results also confirmed the successful delivery of celecoxib. It is important to note that tendon adhesion and tendon healing are two coexisting phenomena in the injury repair process. While studying the prevention of Achilles tendon adhesion, the issue of Achilles tendon healing should not be ignored, as collagen deposition is crucial for Achilles tendon repair. In vivo Achilles tendon tissue and in vitro fibroblasts were stained with Col-1 and COL-III immunofluorescence staining. Figure 14 CD section Figure 14 The middle GH section and Figure 18 , Figure 19 , Figure 20 As shown, there was no statistically significant difference in tendon collagen strength among the groups, indicating that successful celecoxib delivery does not affect the tendon healing process, which may accelerate the maturation and cross-linking of tendon collagen. Therefore, the ultrasound nanobubble coupling agent not only enables real-time ultrasound imaging but also achieves deep, non-invasive drug delivery through the cavitation effect of amplified ultrasound, bringing possibilities for non-invasive treatment of Achilles tendon adhesions, anesthesia and analgesia, tennis elbow, and other diseases.

[0117] III. Conclusion

[0118] This invention constructs an ultrasonic nanobubble coupling agent that, through a cascade rocket-assisted amplification of the ultrasonic cavitation effect, effectively penetrates the skin's dual barriers to achieve deep, non-invasive drug delivery. The ultrasonic coupling agent possesses excellent adhesion, biocompatibility, self-healing properties, and injectability, and can non-destructively transmit ultrasonic energy, cascading to enhance the ultrasonic cavitation effect. In porcine in vivo skin experiments, the effective penetration depth reached 728 μm, and the transdermal drug delivery efficiency was 2.17 times that of traditional coupling agents. A rat Achilles tendon adhesion model was successfully established, with the Achilles tendon adhesion score reduced by 3.2 times compared to the blank control group. Notably, the ultrasonic nanobubble coupling agent not only achieves non-invasive deep drug delivery but also enables non-destructive imaging of the target area, integrating diagnosis and treatment. In summary, the cascade rocket-assisted amplification of the ultrasonic cavitation effect achieved by the ultrasonic nanobubble coupling agent enables non-invasive deep drug delivery, opening a new chapter for non-invasive drug delivery platforms and disease treatment.

[0119] Comparative Example 1

[0120] The drug permeability of the ultrasonic microbubbles described in Example 7 of Patent Document CN 1943541 A was investigated according to the method of the present invention. The results showed that in an in vitro model of isolated pig skin, when the ultrasound parameters were set to 2 W, 650 kHz, 50% duty cycle and ultrasound for 20 min, the effective penetration depth of the drug by the ultrasonic microbubbles was only 302 μm.

[0121] When the ultrasound time is extended to 30 min, the ultrasonic microbubble can achieve an effective drug penetration depth of 328 μm.

[0122] Comparative Example 2

[0123] The drug permeability of the microbubble coupling agent provided in Example 1 of Patent Document CN 105435224 A was investigated according to the method of the present invention. The results showed that in an in vitro model of isolated pig skin, when the ultrasound parameters were set to 2 W, 650 kHz, 50% duty cycle and ultrasound time of 20 min, the effective penetration depth of the drug by the ultrasound microbubble was only 348 μm.

[0124] When the ultrasound time is extended to 30 min, the ultrasonic microbubble can achieve an effective drug penetration depth of 361 μm.

Claims

1. A method for preparing an ultrasonic nanobubble coupling agent, characterized in that, Includes the following steps: (1) Using aminated PLGA, celecoxib and dichloromethane as the oil phase and polyvinyl alcohol and perfluorohexane as the aqueous phase, celecoxib-loaded nanobubbles were obtained by two ultrasonic treatments using a double emulsion method, denoted as Cel@PFH@PLGA-NH2NBs; (2) Hyaluronic acid was aldehyde-modified and amino-modified to prepare HA-CHO and HA-NH2. HA-CHO and HA-NH2 were mixed and gelled to prepare HA-CHO / HA-NH2 hydrogel. (3) The Cel@PFH@PLGA-NH2 NBs were mixed with the HA-CHO / HA-NH2 hydrogel solution and subjected to Schiff base reaction to prepare ultrasonic celecoxib hyaluronic acid hydrogel, which is the ultrasonic nanobubble coupling agent.

2. The preparation method according to claim 1, characterized in that, In step (1), the weight ratio of aminated PLGA to celecoxib is 5:

1.

3. The preparation method according to claim 1, characterized in that, The polyvinyl alcohol in step (1) has a mass-volume concentration of 1% in the aqueous phase, w / v.

4. The preparation method according to claim 1, characterized in that, The preparation method of HA-CHO in step (2) is as follows: hyaluronic acid and sodium periodate are dissolved in water, stirred at 25°C for 4 hours in the dark, the reaction is quenched with ethylene glycol, the solution is dialyzed, and the product is freeze-dried to obtain the final product.

5. The preparation method according to claim 1, characterized in that, The preparation method of HA-NH2 in step (2) is as follows: Hyaluronic acid is dissolved in water, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are dissolved in a mixed solution of dimethyl sulfoxide and water at a volume ratio of 1:1, and then mixed with the hyaluronic acid solution and reacted for 1 hour. Dihydrazine adipate is added, the pH is adjusted to 6.8, and dialyzed. The obtained solution is centrifuged, the supernatant is dialyzed at room temperature, and the obtained product is freeze-dried to obtain the final product.

6. The preparation method according to claim 1, characterized in that, The mass ratio of HA-NH2 and HA-CHO in step (2) is 2:

5.

7. The preparation method according to claim 1, characterized in that, The volume ratio of Cel@PFH@PLGA-NH2 NBs to HA-CHO / HA-NH2 hydrogel in step (3) is 2:

1.

8. The preparation method according to claim 1, characterized in that, The Schiff base reaction time in step (3) is 10 min.

9. An ultrasonic nanobubble coupling agent prepared by the method according to any one of claims 1-8.

10. The use of an ultrasonic nanobubble coupling agent prepared by any one of claims 1-8 or the ultrasonic nanobubble coupling agent as described in claim 9 in the preparation of a drug carrier for non-invasive drug delivery.

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