Ultrasonic response type hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ultrasonic response type hydrogel as well as a preparation method and application thereof. According to the ultrasonic response type hydrogel preparation, an ultrasonic triggering threshold value is reduced to 1W / cm < 2 > by utilizing a synergistic anti-inflammatory mechanism that the Mxene enzyme activity is enhanced by ultrasound, muscle heat tolerance is met, and heat loss is avoided. And under the ultrasonic activation condition, the activity of various enzymes of Mxene is increased. Besides, according to the anti-aging multi-mechanism collaborative design, aiming at inflammation-aging-atrophy vicious circle of sarcopenia, ultrasonic enhanced enzyme activity, anti-inflammation, anti-oxidation and anti-aging treatment of sarcopenia is realized for the first time.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanotechnology and biomedicine, and more specifically, to an ultrasound-responsive hydrogel, its preparation method, and its applications. Background Technology

[0002] Statistics show that the prevalence of sarcopenia in people over 60 years old in my country is 14.7%, which leads to falls, fractures, and multiple organ failure, significantly increasing the medical burden. [1] Current clinical treatments suffer from high 5-year relapse rates due to poor adherence among elderly patients and low muscle repair efficiency. More seriously, sarcopenia is often accompanied by chronic inflammation, which easily leads to muscle atrophy; existing therapies struggle to overcome this pathological bottleneck. [2] .

[0003] Hydrogels, as biomimetic drug delivery systems, have demonstrated unique advantages in the treatment of inflammation and have become an important research direction in the field of musculoskeletal diseases. These hydrophilic three-dimensional polymer materials, by loading traditional anti-inflammatory drugs or bioactive molecules, form local drug reservoirs in the inflammatory microenvironment such as arthritis lesions and postoperative infection sites. [3] Ultrasound is non-invasive, has strong tissue penetration, and is highly spatiotemporally controllable. It can trigger drug release from hydrogels through cavitation, mechanical vibration, or thermal effects. Ultrasound-responsive hydrogels hold promise for enabling sustained drug release within the tissue microenvironment, reducing the frequency of drug administration and lowering systemic toxicity.

[0004] In recent years, the two-dimensional nanomaterial Mxene has provided new insights for upgrading the functionality of hydrogels due to its excellent electrical conductivity. Previous studies have confirmed that Mxene / silk fibroin composite hydrogels can generate mechanical stress waves and electrical stimulation through ultrasonic excitation, promoting Ca2+ metabolism. 2+ Influx activates the CAMKII pathway, accelerating osteoblast differentiation. [4] More importantly, the titanium hydroxyl groups on the surface of Mxene can mimic the activities of various enzymes, specifically scavenging excess reactive oxygen species (ROS) in the inflammatory microenvironment, and hold promise for alleviating various inflammatory diseases. [5] .

[0005] While Mxene-based ultrasound-responsive hydrogels have been used for osteogenic regeneration, their ultrasound-enhanced enzyme activation combined with electrical stimulation has not yet been applied to the treatment of sarcopenia. Furthermore, existing technologies focus on tumor treatment and antibacterial therapy, and no ultrasound-responsive multifunctional hydrogels with anti-inflammatory, anti-aging, and repair-promoting properties have been found specifically for sarcopenia.

[0006] In addition, the trigger threshold for traditional ultrasound response is relatively high (>1.5W / cm). 2Exceeding the heat tolerance limit of muscle tissue can easily induce muscle cell apoptosis. Furthermore, its lack of viscosity and mechanical adaptability means that the hydrogel structure is easily disrupted during exercise, leading to sudden drug release and systemic toxicity.

[0007] Therefore, a device that uses ultrasound to trigger Ca 2+ A multifunctional hydrogel that infiltrates and enhances the anti-inflammatory activity of Mxene multi-enzyme is proposed for the treatment of sarcopenia, and is expected to break through the bottleneck of traditional hydrogels in the treatment of sarcopenia.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide an anti-inflammatory and anti-aging hydrogel formulation based on ultrasound-responsive enhancement of Mxene enzyme activity, its preparation method, and its application.

[0010] In a first aspect of the present invention, a method for preparing MPT@PP hydrogel is provided, the method comprising the steps of:

[0011] (1) Provide an MPT mixture, which is prepared from MXene, PEDOT:PSS (PP) and tannic acid (TA); wherein,

[0012] (2) Provide a mixture P1 and a mixture P2 for forming a hydrogel, wherein mixture P1 is prepared by dispersing PEG-8SH into the MPT mixture in (1) above; and mixture P2 is prepared by dispersing PEG-2Mal into the MPT mixture in (1) above.

[0013] (3) Mix equal volumes of mixture P1 and mixture P2 from (2) to prepare MPT@PP hydrogel;

[0014] (4) Optionally, purification steps for the MPT@PP hydrogel.

[0015] In another preferred embodiment, the concentration of the tannic acid (TA) is 0.1wt% to 0.5wt%, preferably 0.2wt% to 0.4wt%, more preferably 0.25wt% to 0.35wt%, and most preferably 0.3wt%.

[0016] In another preferred embodiment, the MXene is a multilayer MXene.

[0017] In another preferred embodiment, step (1) of preparing the MPT mixture includes the following steps:

[0018] (i) MXene (preferably multilayer MXene) and tannic acid TA are dispersed in a PEDOT:PSS(PP) suspension to obtain a dispersion;

[0019] (ii) The dispersion prepared in (i) above is magnetically stirred for 0.5h to 8h, preferably 1h to 6h, more preferably 3h to 5h, and most preferably 2h to 4h (e.g. 2h);

[0020] (iii) Adjust the pH of the dispersion after magnetic stirring in (ii) above to 6.0-8.0, preferably 6.3-7.8, more preferably 6.5-7.5, and most preferably 7.35-7.45 (e.g. 7.4) to obtain the MPT mixture.

[0021] In a second aspect of the invention, an MPT@PP hydrogel is provided, the hydrogel being prepared by the method described in the first aspect of the invention.

[0022] In another preferred embodiment, the MPT@PP hydrogel is an ultrasonically responsive hydrogel.

[0023] In another preferred embodiment, the MPT@PP hydrogel is an anti-inflammatory hydrogel.

[0024] In another preferred embodiment, the MPT@PP hydrogel is an anti-aging hydrogel.

[0025] In another preferred embodiment, the MPT@PP hydrogel is a conductive, anti-inflammatory, and anti-aging hydrogel.

[0026] In another preferred embodiment, the MPT@PP hydrogel is an ultrasound-responsive conductive anti-inflammatory and anti-aging hydrogel.

[0027] In another preferred embodiment, the tannic acid was purchased from Maclean's, catalog number T818845.

[0028] In a third aspect of the invention, the use of MPT@PP hydrogel in the preparation of medicaments for treating and improving muscle inflammation is provided.

[0029] In a fourth aspect of the invention, the use of MPT@PP hydrogel in the preparation of medicaments for treating and / or preventing muscle aging is provided.

[0030] In a fifth aspect of the invention, the use of MPT@PP hydrogel in the preparation of medicaments for treating and / or improving sarcopenia is provided.

[0031] In another preferred embodiment, the MPT@PP hydrogel is prepared from MXene, tannic acid, PEDOT:PSS(PP), PEG-8SH, and PEG-2Mal.

[0032] In another preferred embodiment, the MPT@PP hydrogel comprises ultrasound-enhanced multilayer Mxene enzyme activity.

[0033] In another preferred embodiment, the MPT@PP hydrogel is an injectable ultrasound-responsive hydrogel for the treatment of sarcopenia.

[0034] In another preferred embodiment, the MPT@PP hydrogel is the MPT@PP hydrogel described in the second aspect of the present invention.

[0035] In another preferred embodiment, the type of sarcopenia is selected from the group consisting of: primary (aging) sarcopenia and drug-induced (cyclophosphamide) sarcopenia.

[0036] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0037] (i) MPT@PP hydrogel; and

[0038] (ii) Pharmaceutically acceptable carriers.

[0039] In another preferred embodiment, the MPT@PP hydrogel is prepared from MXene, tannic acid, PEDOT:PSS(PP), PEG-8SH, and PEG-2Mal.

[0040] In another preferred embodiment, the MPT@PP hydrogel is the MPT@PP hydrogel described in the second aspect of the present invention.

[0041] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating sarcopenia.

[0042] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0043] In another preferred embodiment, the drug for treating sarcopenia is an ultrasound-assisted therapy drug.

[0044] In another preferred embodiment, the carrier is selected from the group consisting of saline, buffer solution, glucose, water, glycerol, ethanol, or combinations thereof.

[0045] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of: intramuscular injection, subcutaneous injection, and intradermal injection.

[0046] In a seventh aspect of the invention, a method for treating sarcopenia is provided, the method using the pharmaceutical composition described in the sixth aspect of the invention.

[0047] In another preferred embodiment, the method is performed with ultrasound assistance.

[0048] In another preferred embodiment, the ultrasound assistance provides ≤1.5 W / cm².2 (Preferred concentration: 0.5–1.2 W / cm) 2 More preferably 0.75~1W / cm 2 Ultrasonic activation.

[0049] In another preferred embodiment, the site of ultrasound activation is the site where the subject is administered the pharmaceutical composition according to the sixth aspect of the invention.

[0050] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0051] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0052] Figure 1 In the diagram, a is a schematic diagram of the biocompatibility click chemistry reaction between thiols and maleimide; b is a schematic diagram of the competitive binding of tannic acid (TA) and thiols; c is a flowchart of the preparation of the ultrasound-responsive hydrogel formulation in Example 1.

[0053] Figure 2 This is a test diagram of the adhesion and stability of the hydrogel under physiological conditions in Example 2;

[0054] Figure 3 Example 3: Safety testing of hydrogel cells;

[0055] Figure 4 Example 4: Ultrasound-enhanced detection of Mxene-like enzyme activity;

[0056] Figure 5 Example 5: In vitro ROS scavenging ability test;

[0057] Figure 6 Example 6: Detection of intracellular ROS scavenging ability;

[0058] Figure 7 Example 7: Detection of intracellular anti-aging ability;

[0059] Figure 8 Safety assessment of each treatment group in the acute sarcopenia model of Example 8;

[0060] Figure 9 This is an evaluation of the efficacy of each treatment group in the acute sarcopenia model of Example 8;

[0061] Figure 10 Example 9: In vivo muscle tissue adhesion ability test. Detailed Implementation

[0062] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0063] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0064] Through extensive and in-depth research, the inventors have addressed the clinical needs and shortcomings of existing technologies in the treatment of sarcopenia. They have designed and prepared for the first time an ultrasound-responsive anti-inflammatory hydrogel formulation that enhances Mxene enzyme activity, primarily for the treatment of sarcopenia.

[0065] This invention belongs to the field of ultrasound-responsive hydrogel technology. Addressing the limitations of existing Mxene hydrogels for focused osteogenic regeneration and tumor treatment, it is the first to construct a multifunctional ultrasound-responsive conductive hydrogel system with anti-inflammatory, anti-aging, and repair-promoting properties for the treatment of sarcopenia. Furthermore, it addresses the issue of the traditional high ultrasound response threshold (>1.5 W / cm²). 2 To address the safety issues, this invention reduces the trigger threshold to 1W / cm². 2 This also reduces muscle cell death. Multiple network cross-linking technologies enhance the adhesion strength of the hydrogel, allowing it to better adhere to the muscle tissue interface.

[0066] Specifically, this invention addresses the application gap and safety deficiencies of ultrasound-responsive conductive anti-inflammatory and anti-aging hydrogels in the treatment of sarcopenia. This invention provides an ultrasound-enhanced Mxene enzyme activity hydrogel formulation, MPT@PP. By integrating Mxene, tannic acid, and various polyethylene glycols (PEGs), a hydrogel system with adhesiveness, stability, and safety is constructed. Experiments have demonstrated that this formulation exhibits good adhesion at a muscle heat tolerance level of 1 W / cm². 2 Under ultrasound, the activity of Mxene-like enzymes is enhanced, and ROS scavenging shows good effects both in vitro and in myoblasts, leading to a reduction in cellular senescence caused by oxidative stress and promoting the activation of calcium ion channels in myoblasts. This invention represents the first successful implementation of ultrasound-enhanced enzyme activity for synergistic anti-inflammatory, anti-aging, and repair-promoting therapy, overcoming the functional limitations of traditional hydrogels in the field of sarcopenia, and demonstrating excellent biocompatibility. Based on this, the invention was completed.

[0067] the term

[0068] As used in this invention, the terms “containing,” “comprising,” or “having” include “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “comprising.”

[0069] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0070] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally, the purification step of the MPT@PP hydrogel” means that the “purification step of the MPT@PP hydrogel” may be present but is not required.

[0071] In this invention, "PEDOT:PSS" stands for Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), and PP is an abbreviation for PEDOT:PSS.

[0072] In this invention, the "MPT mixture" is prepared from MXene, PEDOT:PSS, and tannic acid. Typically, the "MPT mixture" contains 4 wt% multilayer MXene and 0.3 wt% TA in PEDOT:PSS.

[0073] In this invention, "MPT@PEG-8SH" refers to mixture P1 prepared by dispersing PEG-8SH in MPT mixture; "MPT@PEG-2Mal" refers to mixture P2 prepared by dispersing PEG-2Mal in MPT mixture.

[0074] MPT@PP hydrogel

[0075] Molecular modulators (TAs) are introduced during hydrogel network formation to fine-tune reaction kinetics. Typically, this is based on biocompatible click chemistry between thiols (PEG-8SH in this paper) and maleimides (PEG-2Mal in this paper). Figure 1 In study a), it was found that tannic acid (TA), which is rich in phenolic groups, can competitively bind with thiols. Figure 1In b), and effectively regulate gel kinetics to achieve optimal injectability while maintaining excellent mechanical stability.

[0076] The strong covalent bond formed between thiol (-SH) and maleimide (-Mal) via click chemistry ensures the stability of the hydrogel, but their reaction kinetics are very fast. For example, at physiological pH ~7, the gelation time between 8-arm peg-thiol (PEG-8SH) and polymaleimide-polymaleimide (PEG-2Mal) is less than 10 s, making this system unsuitable for injection. Therefore, when designing the hydrogel, TA was introduced as a molecular regulator to slow down the reaction kinetics of the -SH group click chemistry at neutral pH (i.e., to reduce the hydrogel formation time). Its abundant phenolic groups can competitively react with the -SH in PEG-8SH, thus making the gelation time adjustable. Therefore, ICAA hydrogels can be easily injected into any random surface as a homogeneous mixture, maintaining stable conformal and intimate contact even under large deformations.

[0077] In addition, TA can enhance the multi-scale hydrogen bonding interactions between micron-scale conductive MXene and nano-scale conductive polymer PEDOT:PSS, thereby enhancing conductivity.

[0078] Self-assembled MXene and PEDOT:PSS were used as conductive additives, with MXene forming a layered structure (~25 μm) and PEDOT:PSS forming an aqueous colloidal suspension (~100 nm). Both materials can be stacked and entangled into conductive networks through π-π stacking and hydrogen bonding interactions. Furthermore, TA can act as a molecular modifier to introduce abundant hydrogen bonding interactions, promoting π-π stacking between PEDOT+ chains and MXene. Therefore, the non-covalent multi-scale conductive network strategy enhances conductivity compared to a simple mixture of MXene and PEDOT:PSS.

[0079] Furthermore, the presence of tannins as a bridge between the hydrogel matrix and the conductive network gives the hydrogel excellent anti-swelling properties. This is due to its higher morphological roughness and greater aggregation and uniformity.

[0080] Finally, the abundant cross-linking chemicals within the network provide immediate bioadhesion. In the sol state, due to the abundance of phenolic groups in TA, the precursor (MPT) forms various dynamic non-covalent interactions, such as hydrogen bonds, through rapid adsorption and removal of interfacial water and conformal interlocking with the contact surface. During the sol-gel transition phase (~60 s), the diffused MPT precursor undergoes continuous -SH group click reactions at the hydrogel-substrate or hydrogel-tissue interface. This reaction is the main driving force, firmly anchoring the MPT@PP hydrogel between the wet device substrate and the tissue.

[0081] Therefore, MPT@PP hydrogels with stable irreversible covalent PEG networks and multi-scale non-covalent conductive networks exhibit significantly improved electrochemical and tissue-like mechanical properties.

[0082] In this invention, the MPT@PP hydrogel was prepared from MXene, tannic acid, PEDOT:PSS(PP), PEG-8SH, and PEG-2Mal.

[0083] For example, the preparation method of MPT@PP hydrogel can be found in Nature Communications (2024) 15: 7993.

[0084] In a specific embodiment of the present invention, the flowchart of the hydrogel formulation is shown below. Figure 1 c.

[0085] In a specific embodiment of the present invention, the preparation method of MPT@PP hydrogel is as follows:

[0086] 80 mg of multilayer titanium carbide (clay-like) (MXene) and 6 mg of tannic acid (TA) were dispersed in 2 g of PEDOT:PSS (PP) suspension. After magnetic stirring for 2 h, the pH was adjusted to 7.4 to obtain MPT mixture (PEDOT:PSS dispersion system containing 4 wt% multilayer MXene and 0.3 wt% TA).

[0087] 176.5 mg of PEG-8SH powder and 111 mg of PEG-2Mal powder were dissolved in 1 g of MPT dispersion system (PEDOT:PSS dispersion system containing 4 wt% multilayer Mxene and 0.3 wt% TA, after pH adjustment, i.e., MPT mixture) to form 15 wt% MPT@PEG-8SH and 10 wt% MPT@PEG-2Mal dispersions for later use. The 15 wt% MPT@PEG-8SH and 10 wt% MPT@PEG-2Mal were then mixed in equal volumes to prepare MPT@PP hydrogel.

[0088] sarcopenia

[0089] Sarcopenia, also known as muscle loss or muscle volume reduction, is an age-related progressive decrease in total muscle mass and / or muscle strength or muscle function. Sarcopenia is closely associated with mobility impairments, falls, low bone density, and metabolic disorders. It is a significant cause and manifestation of the gradual decline in physiological function in older adults and is a common geriatric syndrome.

[0090] Sarcopenia lacks specific clinical manifestations. Patients may experience frequent falls, fractures, difficulty walking, slow gait, thin and weak limbs, and even complications such as bedsores, pneumonia, fractures, and death due to prolonged bed rest caused by mobility impairment.

[0091] Sarcopenia is caused by many internal and external factors, such as decreased hormone levels, reduced physical activity, weakened neuromuscular function, decreased vitamin D intake, pro-inflammatory cytokines, malnutrition, insufficient protein intake, and medications. It can also be secondary to common chronic diseases, such as chronic heart failure, chronic obstructive pulmonary disease, diabetes, chronic kidney disease, connective tissue diseases, tuberculosis infection, and some chronic digestive diseases (malignant tumors, cirrhosis, etc.).

[0092] The diagnosis of sarcopenia includes decreased muscle mass, decreased muscle strength, and decreased muscle function. Muscle mass is assessed using dual-energy X-ray absorptiometry (DXA), CT, and MRI. Muscle strength is most commonly assessed using grip strength testing. Muscle function is assessed using the Simplified Body Function Assessment (SWFA), daily walking speed assessment, stand-up walking test, and stair climbing test. Currently, the European Working Group on Sarcopenia Standards (EWGSOP) are widely used internationally for the diagnosis of sarcopenia. These standards include: walking speed <0.8 m / s; grip strength <30 kg for men and <20 kg for women; and muscle mass (AST / height²) of the limbs: below two standard deviations from the average of the youth control group. Meeting all three criteria is sufficient for a diagnosis of sarcopenia.

[0093] Pharmaceutical Composition

[0094] The present invention also provides a pharmaceutical composition comprising the above-described MPT@PP hydrogel and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intramuscular, intravenous, local, subcutaneous, or intradermal injection.

[0095] The pharmaceutical composition of this invention can be directly applied to muscle tissue to remove excess ROS, achieving anti-inflammatory, antioxidant, and / or anti-aging treatment of muscle tissue. Furthermore, the multi-mechanism synergistic design of this invention targets the vicious cycle of inflammation-aging-atrophy in sarcopenia, achieving ultrasound-enhanced enzyme activation for anti-inflammatory, antioxidant, and anti-aging treatment of sarcopenia. In addition, other therapeutic agents can be used simultaneously.

[0096] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the MPT@PP hydrogel described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 10 milligrams / kg body weight per day. Furthermore, the MPT@PP hydrogel of the present invention can also be used with other therapeutic agents.

[0097] When using the pharmaceutical composition, a safe and effective amount of MPT@PP hydrogel is administered to mammals, wherein this safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0098] The beneficial effects of this invention include:

[0099] 1. By enhancing the activity of Mxene enzyme through ultrasound, the ROS clearance rate is enhanced both in vivo and in vitro, thereby clearing excess ROS caused by oxidative stress in muscle cells, protecting muscle cells from cellular aging caused by oxidative stress, and breaking the vicious cycle of inflammation-aging-atrophy in sarcopenia.

[0100] 2. Good safety profile, ultrasound threshold 1W / cm 2 It is below the heat tolerance level, does not affect cell survival, and meets clinical safety standards.

[0101] 3. Innovative application scenarios: Existing patents focus on tumor and antibacterial treatment. This invention is the first to use ultrasound-responsive hydrogel for anti-inflammatory, antioxidant and anti-aging treatment of sarcopenia. It is the first Mxene-based ultrasound-responsive anti-inflammatory and anti-aging conductive hydrogel for sarcopenia.

[0102] The present invention will be further illustrated below with reference to specific embodiments.

[0103] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0104] Example 1: Preparation of MPT@PP hydrogel

[0105] 80 mg of multilayer titanium carbide MXene (clay-like) (purchased from Foshan Xinxi Technology Co., Ltd., item number 12363-89-2, 400 mesh, specification 1g) and 6 mg of tannic acid TA (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number T818845, specification 10g) were dispersed in 2g of PEDOT:PSS(PP) (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number P758999, specification 25ml) suspension. After magnetic stirring for 2 hours, the pH was adjusted to 7.4 to obtain the MPT mixture. 176.5 mg of PEG-8SH powder (purchased from Shanghai Yanyi Trading Co., Ltd., item number 8276-734-74-20K-1G, specification MW20000) and 111 mg of PEG-2Mal powder (purchased from Shanghai Yanyi Trading Co., Ltd., item number 625-734-625-5K-1G, specification MW5000) were dissolved in 1 g of MPT dispersion system (a PEDOT:PSS dispersion system containing 4 wt% multilayer Mxene and 0.3 wt% TA, pH adjusted, i.e., MPT mixture) to form 15 wt% MPT@PEG-8SH and 10 wt% MPT@PEG-2Mal dispersions for later use. Equal volumes of 15 wt% MPT@PEG-8SH and 10 wt% MPT@PEG-2Mal were then mixed to prepare MPT@PP hydrogel. The flowchart for the specific hydrogel formulation is shown below. Figure 1 c.

[0106] Example 2 Adhesion and Stability Test

[0107] 10 μL of MPT@PEG-8SH and 10 μL of MPT@PEG-2Mal were injected sequentially into the interface of isolated C57BL / 6 fractional leg muscle tissue. After 30-60 seconds for the hydrogels to crosslink and adhere to MPT@PP, the isolated muscle tissue with the MPT@PP hydrogel adhering was immersed in pH 7.4 phosphate buffer and placed in a 37°C incubator to simulate a physiological environment. The results are as follows... Figure 2 As shown. From Figure 2 As can be seen, MPT@PP hydrogel can stably adhere to muscle tissue for 24 hours under physiological conditions.

[0108] Example 3 Cell safety testing

[0109] Sterilized MPT@PP hydrogel was spread at the bottom of a 12-well plate (5 wells were coated with 20 μl of MPT@PP hydrogel as the experimental group, and the other 5 wells were coated with 20 μl of PBS buffer as the control group. After the hydrogel solidified, the plates were sterilized with UV light for 30 minutes. This process was repeated for three wells, creating three different detection time points). Each well in both the control and experimental groups was inoculated with 1 x 10-1 MPT@PP hydrogel. 5 C2C12 cells (derived from muscle tissue of C3H strain mice, purchased from Shanghai Yuanchuang Biotechnology Co., Ltd., catalog number YC-M0237, 1ml / T25) were added to each well, along with 1ml of C2C12 cell culture medium (iCell-m013-001b, 500ml). The cells were cultured at 37℃, 5% CO2, and 90% humidity. On days 1, 3, and 5, 400μl of CCK-8 reagent was added to 3600μL of serum-free C2C12 cell culture medium to prepare the CCK-8 detection working solution, which was prepared fresh and used immediately in the dark. After aspirating the liquid from the 12-well plate and washing twice with PBS, 300ul of the prepared CCK-8 detection working solution was added to each well. The plate was incubated for 30 minutes in the dark. 100ul of the supernatant was then transferred to a 96-well plate, and the absorbance was measured at 450nm using a microplate reader to assess cell safety. The results for the experimental and control groups are as follows. Figure 3 As shown, there was no statistically significant difference (ns) in cell viability between the experimental group and the control group as the co-incubation time increased, demonstrating the safety of using MPT@PP hydrogel for cells.

[0110] Example 4: Detection of Mxene-like enzyme activity enhanced by ultrasound

[0111] 0.1624 g of 2,2'-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number A800764, 1 g) was dissolved in 40 ml of deionized water to prepare a 7.4 mmol / L ABTS solution. 0.0281 g of potassium persulfate (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number P816371, 100 g) was dissolved in 40 ml of water to prepare a 2.6 mmol / L potassium persulfate solution. 2 ml of the ABTS solution (7.4 mmol / L) and 2 ml of the potassium persulfate solution (2.6 mmol / L) were mixed and reacted in the dark for 24 hours to generate ABTS-+. The solution was diluted 50 times with phosphate buffer (normal buffer, pH 7.4) to prepare the working solution (completely dissolved). 1 mL of solutions of PP, tannic acid, Mxene, and MPT of equal concentration were mixed with 1 mL of ABTS-+ solution and reacted for 10 minutes. The UV-Vis spectra were then measured at 734 nm. The sonication conditions were 1 W / cm² after solution mixing. 2 Duty cycle 50%, 1 minute. Detection results as follows: Figure 4 As shown, under the action of ultrasound, the enzyme activity of Mxene is enhanced and the absorbance is lower, and the ability of ultrasound to enhance the enzyme activity of Mxene is retained in MPT.

[0112] Example 5: In vitro ROS scavenging ability test

[0113] Weigh 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number D807297, specification 100mg), dissolve in anhydrous ethanol to prepare a 0.1 mmol / L solution (stored protected from light). Dilute the hydrogel preparation MPT tenfold to obtain concentration 1, and then perform serial dilutions to obtain sample solutions of different concentrations. Take 200 μL of the sample solution, add 200 μL of DPPH solution (0.1 mmol / L), mix well, and react at room temperature in the dark for 30 minutes. Control group: 200 μL water + 200 μL DPPH solution (0.1 mmol / L); Blank group: 200 μL sample solution + 200 μL water. Measure the absorbance at a wavelength of 517 nm using a spectrophotometer. Scavenging rate (%) = (1 - ((sample A - blank A) / control A)) × 100%. Results are as follows. Figure 5 As shown, the scavenging effect of DPPH free radicals increases with increasing MPT concentration, exhibiting a concentration-dependent relationship.

[0114] Example 6: Detection of Intracellular ROS Scavenging Capacity

[0115] C2C12 mouse myoblasts (derived from muscle tissue of C3H strain mice, purchased from Shanghai Yuanchuang Biotechnology Co., Ltd., catalog number YC-M0237, specification 1ml / T25) were seeded in 12-well plates and cultured for 24 hours at 37℃, 5% CO2, and 90% humidity. Hydrogen peroxide (H2O2) at 200μM was then added to the cell culture medium, and the cells were incubated at 37℃ for 24 hours to simulate inflammatory oxidative stress. MPT@PP hydrogel was placed in Transwell chambers and co-incubated with the cells for 4 hours. For the ultrasound group: the culture medium was aspirated from the cell plate, and the ultrasound probe was coated with coupling agent and placed close to the lower wall of the cell plate for ultrasound at 1W / cm². 2 The cells were sonicated for 1 minute at a 50% duty cycle, followed by the addition of culture medium. The control group underwent only medium replacement without sonication. Intracellular ROS levels were detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0033S, reactive oxygen species detection kit). DCFH-DA was prepared as a working solution in serum-free medium to a final concentration of 10 μM and added to the cells. The cells were incubated at 37°C in the dark for 20 minutes. The cells were washed three times with serum-free medium to remove the free probe. The fluorescence distribution of DCFH-DA (green fluorescence) was observed under a fluorescence microscope. Results are as follows: Figure 6 As shown, MPT@PP hydrogel can significantly reduce intracellular ROS levels, but cannot completely eliminate them. However, under ultrasound-enhanced enzyme activity, MPT@PP hydrogel can almost completely eliminate residual ROS in cells.

[0116] Example 7: Detection of Intracellular Anti-aging Capacity

[0117] C2C12 mouse myoblasts were seeded in 12-well plates and cultured for 24 hours at 37°C, 5% CO2, and 90% humidity. Then, they were induced to the myotube state for 5 days with 10% horse serum (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number 26050070, 100ml). Sodium palmitate (200μM) was added and incubated at 37°C for 3 days until cellular senescence following inflammatory stress. MPT@PP hydrogel was placed in Transwell chambers and co-incubated with the cells for 24 hours. For the ultrasound group: the culture medium was aspirated from the cell plate, and the ultrasound probe was coated with coupling agent and placed close to the lower wall of the cell plate for ultrasound at 1W / cm². 2The cells were sonicated for 1 minute at a 50% duty cycle, followed by the addition of culture medium. Control group: only the culture medium was changed, without sonication. The culture medium was aspirated, and the cells were washed twice with phosphate-buffered saline (PBS). 1 mL of fixative (Gluta fixative (electron microscopy, 2.5), Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and the cells were fixed at room temperature for 15 minutes. The fixative was aspirated, and the cells were washed three times with phosphate-buffered saline (conventional PBS buffer, phosphate-buffered saline, pH 7.4, Thermo Fisher Scientific (China) Co., Ltd., 10010023) for 5 minutes each time. Following the instructions of the Cell Senescence β-Galactosidase Staining Kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0602), staining solutions A, B, C, and X-Gal were mixed in the proportions shown in the instructions to prepare the working staining solution. 1 mL of the working staining solution was added to each well, and the cells were incubated overnight at 37°C (CO2-free). The working staining solution was then removed, 2 mL of PBS was added, and the cells were observed using an optical microscope. Randomly selected fields of view were photographed. Results are as follows: Figure 7 As shown, the MPT@PP+ultrasound group had the lowest percentage of senescent cells, indicating that ultrasound enhanced the anti-aging effect of MPT@PP.

[0118] Example 8: Evaluation of the therapeutic effect of an acute sarcopenia model

[0119] Twenty-five 6-8 week old female C57BL / 6 mice (Shanghai Kelings Biotechnology Co., Ltd.) were randomly divided into 5 groups (n=5 per group): a healthy group, a control group, an ultrasound group, an MPT@PP group, and an MPT@PP+US group. Except for the healthy group, mice in the other groups were injected intramuscularly with 100 μL (10 μM) of cardiotoxin (CTX) IV(6-12) TFA per mouse to establish an acute sarcopenia mouse model. On the first day of treatment, MPT@PP was injected into the surface of the tibialis anterior muscle of mice in the MPT@PP and MPT@PP+US groups. Every two days, the tibialis anterior muscle of mice in the MPT@PP+US group was subjected to ultrasound therapy at a rate of 1 W / cm². 2 The treatment duration was 50% duty cycle and 3 minutes. The treatment lasted 14 days. Weight changes in each group were monitored to assess the biocompatibility of the treatment, and final muscle strength and muscle mass were measured to assess efficacy. Results showed no significant difference in weight among the groups, indicating that MPT@PP+US treatment has good safety. Figure 8 ). Figure 9 The results of muscle strength and muscle weight showed that MPT@PP+US treatment could effectively reverse muscle damage caused by acute inflammation and effectively promote the regeneration and repair of muscle strength and muscle mass in mice.

[0120] Example 9: Detection of Muscle Tissue Adhesion Ability in Aged Mice

[0121] Fifteen-month-old female C57BL / 6 mice were selected as aged sarcopenia models. MPT@PP hydrogel was injected into the surface of the tibialis anterior muscle of the mice, and the adhesion of the hydrogel to the surface of the muscle of the live mice was detected on days 0, 7, and 28. Figure 10 The results showed that after injection, MPT@PP hydrogel adhered well to the surface of the tibialis anterior muscle in mice, forming a hydrogel. Furthermore, it maintained good adhesion at the injection site on days 7 and 28 after the mice resumed normal activity. These results indicate that MPT@PP has excellent tissue adhesion ability in the muscle tissue of aged mice and can maintain adhesion even after the mice resume normal activity.

[0122] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

[0123] References

[0124] 1. Chinese Medical Association Geriatrics Branch, National Clinical Research Center for Geriatric Diseases (Xiangya Hospital), Chinese Medical Journal, 2025, 105, 181.

[0125] 2.ABSVasconcelos,AGdeResende-Neto,ACNogueira,JC MRPMonteiro,GSMorais Junior,GGAvelar,EACamargo,O.deT. MEDa Silva-Grigoletto,Exp.Gerontol.2020,135,110920.

[0126] 3.Y.Sun,R.Sheng,Z.Cao,C.Liu,J.Li,P.Zhang,Y.Du,Q.Mo,Q.Yao,J.Chen,W.Zhang,Sci.Adv.2024,10,eadm7164.

[0127] 4.Z.-C.Hu,J.-Q.Lu,T.-W.Zhang,H.-F.Liang,H.Yuan,D.-H.Su,W.Ding,R.-X.Lian,Y.-X.Ge,B.Liang,J.Dong,X.-G.Zhou,L.-B.Jiang,Bioact.Mater.2023,22,1.

[0128] 5.W.Feng,X.Han,H.Hu,M.Chang,L.Ding,H.Xiang,Y.Chen,Y.Li,Nat.Commun.2021,12,2203.

Claims

1. A method for preparing MPT@PP hydrogel, characterized in that, The method includes the following steps: (1) Provide an MPT mixture, which is prepared from MXene, PEDOT:PSS and tannic acid; wherein, (2) Provide a mixture P1 and a mixture P2 for forming a hydrogel, wherein mixture P1 is prepared by dispersing PEG-8SH into the MPT mixture in (1) above; and mixture P2 is prepared by dispersing PEG-2Mal into the MPT mixture in (1) above. (3) Mix equal volumes of mixture P1 and mixture P2 from (2) to prepare MPT@PP hydrogel; (4) Optionally, purification steps for the MPT@PP hydrogel.

2. The preparation method according to claim 1, characterized in that, The concentration of the tannic acid is 0.1 wt% to 0.5 wt%.

3. The preparation method according to claim 1, characterized in that, In step (1), the preparation of the MPT mixture includes the following steps: (i) MXene and TA tannin were dispersed in a PEDOT:PSS suspension to obtain a dispersion; (ii) The dispersion prepared in (i) above is magnetically stirred for 0.5 h to 8 h; (iii) Adjust the pH of the dispersion after magnetic stirring in (ii) above to 6.0-8.0 to obtain the MPT mixture.

4. An MPT@PP hydrogel, characterized in that, The hydrogel is prepared by the method described in any one of claims 1 to 3.

5. Application of MPT@PP hydrogel in the preparation of drugs for treating and / or improving sarcopenia.

6. The application as described in claim 5, characterized in that, The types of sarcopenia are selected from the following group: primary sarcopenia and drug-induced sarcopenia.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) MPT@PP hydrogel; and (ii) Pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is used to prepare a medicament for treating sarcopenia.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an injectable dosage form.

10. The pharmaceutical composition according to claim 8, characterized in that, The medication used to treat sarcopenia is an ultrasound-assisted therapy medication.

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