A method for preparing a drug-controlled release in-situ nanocomposite hydrogel

Abstract

The application provides a preparation method of a drug controllable release in-situ nano composite hydrogel, creatively introduces a nano liposome technology, a ROS response component synthesis step and an in-situ nano composite hydrogel formation step under ultrasonic control, various steps are combined, the in-situ nano composite hydrogel is obtained, the preparation method of the obtained hydrogel system can obtain the in-situ nano hydrogel through ultrasonic stimulation under different use environments, and the drug can be controllably released, the preparation method greatly improves the use environment and application range of the nano hydrogel in the field, the drug is added, the ROS generation amount and the sustained release of the drug are realized through the combination of various mechanisms, the precise application of specific drugs is beneficial, and the preparation method has extremely wide industrial application prospects.

Description

Technical Field

[0001] This invention provides a method for preparing an in-situ nanocomposite hydrogel with controlled drug release, belonging to the field of biomaterial preparation technology. Background Technology

[0002] In-situ gels typically exist in a liquid state at room temperature. Upon application to the drug site, they undergo a phase transition, changing from a liquid to a solid gel state in response to physiological conditions or other stimuli. Hydrogels (such as collagen, alginate, and chitosan) have been widely used for defect filling due to their excellent biocompatibility and biodegradability caused by their 3D water network structure. Furthermore, stimulus-responsive hydrogel systems can be classified according to different gelation theories, including temperature-responsive, pH-responsive, chemical-responsive, light-responsive, and enzyme-responsive hydrogels, which have been developed for delivery carriers. For example, Madry et al. prepared a thermosensitive hydrogel based on polyethylene oxide (PEO)-polypropylene oxide (PPO)-PEO poloxamer, achieving controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector and further improving full-thickness cartilage repair by overexpressing the cartilage Sox9 transcription factor. Hu et al. prepared a methacrylamide gelatin (GelMA) / nanoclay hydrogel using UV crosslinking to achieve sustained release of extracellular vesicles for cartilage regeneration. However, all of the above-mentioned responsive hydrogels have drawbacks. For example, temperature-responsive hydrogels mainly depend on the body's temperature (37 ℃), resulting in a narrow gelation temperature range; higher gelation temperatures require external stimulation. pH-sensitive hydrogels are limited by the pH value of the injection site, significantly restricting their applicability. Chemically responsive hydrogels may be toxic. Photoresponsive hydrogels, limited by light penetration into tissues, can only control gelation in the superficial epidermis. Achieving in-situ controlled gelation remains a significant challenge among these strategies. Summary of the Invention

[0003] To address the aforementioned technical problems, the applicant seeks to provide a novel method for preparing in-situ nanocomposite hydrogels. This method combines multiple technologies, including in-situ nanocomposite hydrogel technology, ROS-responsive liposome technology, and acoustic control technology. The aim is to provide a novel gel-forming method accompanied by a physical morphological change. Through ultrasonic control, an in-situ hydrogel is rapidly formed, and this hydrogel simultaneously exhibits a sustained-release function. This patent uses KGN drugs as an example for systematic description.

[0004] Based on the above description, the applicant provides a method for preparing an in-situ nanocomposite hydrogel with controlled drug release, used for the preparation of an in-situ nanocomposite hydrogel system with controlled release of reactive oxygen species, comprising the following steps: 1) Synthesize the liposome precursor DSPE-TK-mPEG with reactive oxygen species response; 2) React the DSPE-TK-mPEG obtained in step 1) with liposome components to prepare nanoliposomes LP with reactive oxygen species response; 3) Prepare the nanoliposome LPDK by loading the lipid-soluble drug D with reactive oxygen species responsive LP in step 2). 4) Prepare reactive oxygen species-responsive liposomes (LPDT) from the LPD in step 3) and load them with thrombin T to obtain nanoliposomes LPDT; 5) Add the LPDT from step 4) to the sound-sensitizing agent, so that the sound-sensitizing agent is embedded inside the LPDT; 6) Mix the LPDT containing the acoustic sensitizer obtained in step 5) with fibrinogen to obtain an in-situ nanocomposite hydrogel precursor solution for controlled release of reactive oxygen species-responsive drugs. 7) Under ultrasound stimulation, the precursor solution obtained in step 6) forms an in-situ nanocomposite hydrogel for controlled release of the drug.

[0005] As can be seen from the above steps, the applicant has combined reactive oxygen species (ROS) response technology, liposome embedding technology, in-situ gel technology, and ultrasonic acoustic control technology in a comprehensive manner.

[0006] Research Approach: 1. A ROS-responsive sensitive component, DSPE-TK-mPEG, was established. This component then established a connection with ultrasound via a sonosensitive agent, followed by the encapsulation of corresponding drugs, such as Kartogenin. 2. Nanoscale liposomes were constructed using the above component, aiming to create an ultrasound-controlled liposome. 3. Simultaneously, to obtain an ultrasound-controlled gel, the components of the in-situ hydrogel were separated. For example, thrombin was placed within the liposomes, while fibrinogen remained outside, establishing a liposome isolation mechanism. Only upon ultrasound stimulation can the liposomes be broken, allowing the in-situ composite nanohydrogel to form and simultaneously initiating the controlled-release drug delivery process. This represents a novel preparation method and gel usage approach.

[0007] Furthermore, in the above-mentioned method for preparing in-situ nanocomposite hydrogels with controlled drug release, the synthesis of DSPE-TK-mPEG employs a three-step strategy, consisting of a ketone thiocondensation TK synthesis step, a TK-mPEG synthesis step, and a DSPE-TK-mPEG synthesis step. The aim is to obtain the ROS-sensitive component DSPE-TK-mPEG.

[0008] Furthermore, in the above-mentioned method for preparing the in-situ nanocomposite hydrogel with controlled drug release, the ketithiothiol TK synthesis step is obtained by reacting mercaptopropionic acid, acetone, and trifluoroacetic acid, followed by crystallization, filtration, and washing. Preferably, the ratio of mercaptopropionic acid, acetone, and trifluoroacetic acid is 5-25 g: 1-55 mg: 10-100 μL.

[0009] Furthermore, in the above-mentioned method for preparing in-situ nanocomposite hydrogels with controlled drug release, the TK-mPEG synthesis step involves reacting ketithiolide (TK), methoxyPEG amino (mPEG), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and NHS at room temperature with stirring, followed by dialysis and filtration to obtain the TK-mPEG. The ratio of the TK-mPEG used is 25-100 mg: 50-350 mg: 100-500 mg: 78-430 mg, and the molecular weight cutoff of the dialysis bag is 1 KWD.

[0010] Furthermore, in the above-mentioned method for preparing in-situ nanocomposite hydrogels with controlled drug release, the DSPE-TK-mPEG synthesis step involves adding distearate phosphatidylethanolamine (DSPE), TK-mPEG, DCC, and NHS to a solvent and catalyst, followed by dialysis to obtain the DSPE-TK-mPEG. The dosage ratio is 15-75 mg: 25-150 mg: 4-40 mg: 2-20 mg, and the molecular weight cutoff of the dialysis bag is 7 KWD.

[0011] Furthermore, in the above-mentioned method for preparing in-situ nanocomposite hydrogels with controlled drug release, the order of steps 2), 3), 4), and 5) can be combined or adjusted. That is, thrombin, lipid-soluble drugs, and sound-sensitive agents can be added together or added in their own order.

[0012] Furthermore, in the above-mentioned method for preparing in-situ nanocomposite hydrogels with controlled drug release, LPD is obtained by thin-film hydration or anhydrous ethanol injection.

[0013] Step 2) The operation method of using thin film hydration or anhydrous ethanol injection is a common method for preparing liposomes.

[0014] The common liposome components used in this article, such as phospholipids, are of types and proportions known in the art.

[0015] Furthermore, in the preparation method of the in-situ nanocomposite hydrogel with controlled drug release, the acoustic sensitizer is selected from one or more of HpD, PpIX, and Ce6. PpIX is preferred.

[0016] The working mechanism is that the sonication agent generates reactive oxygen species (ROS) under ultrasound, and the ROS reacts with TK in DSPE-TK-mPEG to stimulate the rupture of liposomes.

[0017] Furthermore, the weight percentage of the sonosensitive agent relative to the total mass of the liposomes ranges from 0.1% to 0.6%. If the sonosensitive agent percentage is too low, the liposomes will not break down sufficiently; if it is too high, it will damage the cells.

[0018] Furthermore, in the preparation method of the in-situ nanocomposite hydrogel with controlled drug release, the lipid-soluble drug D is selected from Kartogenin or doxorubicin.

[0019] Furthermore, in the preparation method of the in-situ nanocomposite hydrogel with controlled drug release, the lipid-soluble drug D is selected from Kartogenin.

[0020] Furthermore, in the preparation method of the in-situ nanocomposite hydrogel with controlled drug release, the weight of the lipid-soluble drug D accounts for 0.01%-0.8% of the total mass of the liposomes. The dosage of the gel and the drug formulation is adjusted according to the encapsulation efficiency and dosage of the drug to be used.

[0021] Furthermore, in the preparation method of in-situ nanocomposite hydrogels with controlled drug release, the ultrasonic action mechanism of the hydrogel is as follows: the above-mentioned nanoscale drug-loaded liposomes with reactive oxygen species are mixed with fibrinogen in a certain proportion. Under ultrasound, the DSPE-TK-mPEG-based liposomes are destroyed by redox reaction with ROS. Subsequently, thrombin is released from the liposomes and reacts with fibrinogen through enzymatic reaction, realizing the transformation of the hydrogel precursor solution from liquid to gel state to form an in-situ hydrogel.

[0022] Furthermore, in the preparation method of the in-situ nanocomposite hydrogel with controlled drug release, the fibrinogen and thrombin in the liposomes are mixed at a volume ratio of 1:0.5-3, with concentrations of 20-400 mg / ml and 2-1000 UI / ml, respectively. The specific dosage is adjusted according to the gel formation strength and drug loading.

[0023] Furthermore, in the preparation method of in-situ nanocomposite hydrogels with controlled drug release, the ultrasonic power range is 0.5-5 W / cm². 2 The ultrasound time is 1-5 minutes.

[0024] Furthermore, in the preparation method of in-situ nanocomposite hydrogels with controlled drug release, thrombin is released from liposomes under ultrasonic stimulation and can form in-situ hydrogels through enzymatic reaction with fibrinogen, thereby realizing the transformation of the hydrogel precursor solution from liquid phase to solid phase.

[0025] Furthermore, in the preparation method of in-situ nanocomposite hydrogel with controlled drug release, under ultrasonic stimulation, the lipid-soluble drug D is Kargenin KGN. The generation of ROS and the controlled release of the cartilage differentiation-promoting drug (KGN) can activate the Smad5 / mTOR signaling pathway and thus promote cartilage regeneration.

[0026] The applicant further provides an in-situ nanocomposite hydrogel with controlled drug release obtained by the above preparation method.

[0027] Preferably, in the above-mentioned in-situ nanocomposite hydrogel with controlled drug release, the lipid-soluble drug D is Kargenin.

[0028] The present invention further provides an application of the above-mentioned drug-controlled release in-situ nanocomposite hydrogel in the preparation of cartilage repair drugs.

[0029] Taking the lipid-soluble drug Kargenin as an example, the working principle of the preparation method of this invention is as follows: Under ultrasonic stimulation, the action of a sonication agent promotes the cleavage of distearate phosphatidylethanolamine-ketothiol-methoxy-polyethylene glycol, releasing thrombin. Then, fibrinogen and thrombin are gelled in situ through an enzymatic reaction. Furthermore, the level of reactive oxygen species (ROS) and the amount of released KGN are detected by adjusting the ultrasonic conditions.

[0030] To address the challenge of precisely controlling on-demand gelation in situ within the body, the applicant proposes a method for manufacturing an in-situ nanohydrogel system responsive to ultrasonic stimulation of reactive oxygen species (ROS). This method leverages the deep tissue penetration of ultrasound to achieve in-situ responsive stimulation. To realize the theory of in-situ gelation, this paper utilizes ketithiothiol (TK)-based liposomes loaded with the bioactive drug KGN, the bioenzyme thrombin, and a sonication agent (PpIX). Under ultrasonic stimulation, the TK-based liposomes are partially disrupted due to the generation of ROS, releasing thrombin, which then gels in situ through an enzymatic reaction involving fibrinogen and thrombin. Furthermore, the level of generated ROS and the amount of released KGN are monitored by adjusting the ultrasound conditions. In cellular experiments, the in-situ nanocomposite hydrogel of fibrinogen and liposome@PpIX-KGN-thrombin (fibrin hydrogel-liposome@PPIX-KGN-thrombin) exhibited good biocompatibility and cartilage differentiation of BMSCs upon ultrasound stimulation via the Smad5 / mTOR signaling pathway. Overall, this in-situ nanocomposite hydrogel effectively improved tissue regeneration in a rat cartilage defect model by combining sustained KGN release with enhanced reactive oxygen species (ROS) production, paving the way for responsive in-situ hydrogels in the field of regeneration.

[0031] This invention also provides a specific method for preparing an in-situ nanocomposite hydrogel with controlled drug release, comprising the following steps: 1) Synthesize DSPE-TK-mPEG by reacting mercaptopropionic acid, acetone, and trifluoroacetic acid, followed by crystallization, filtration, and washing to obtain ketithiothiol (TK). Then, ketithiothiol (TK), methoxyPEG amino (m-PEG), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are stirred and reacted, dialyzed, and filtered to obtain TK-m-PEG. Distearate phosphatidylethanolamine (DSPE) is added, and DSPE-TK-mPEG is obtained under the action of solvent and catalyst. 2) Dissolve phospholipids and DSPE-TK-mPEG in a solvent. Then, dissolve the sound-sensitizing agent and the lipid-soluble drug D separately in the solvent. After mixing the two solutions, rotary evaporate to obtain a phospholipid membrane containing the sound-sensitizing agent, the lipid-soluble drug, and DSPE-TK-mPEG. Dissolve the membrane in water and perform membrane hydration to obtain a mixed solution. Add thrombin and stir under ice bath conditions. Control the size using membrane extrusion to obtain nanoscale liposomes. 3) The above-mentioned nano-sized liposomes are mixed with fibrinogen, and the hydrogel precursor solution is transformed from liquid to solid under ultrasound to obtain the in-situ nanocomposite hydrogel containing the drug for controlled release.

[0032] In the above method parameters, the solvent and catalyst are technologies well known to those in the art.

[0033] This system fully utilizes the response mechanism of DSPE-TK-mPEG and ROS. During use, the above-mentioned ROS-responsive in-situ nanohydrogel is delivered to the relevant site. Through local ultrasound treatment, the sonosensitive agent in the liposome reacts and generates ROS. DSPE-TK-mPEG then breaks down, thereby destroying the liposome and promoting the release of thrombin and lipid-soluble drugs. Thrombin reacts with fibrinogen to obtain a water-soluble gel, which continues to control the slow release of the drug. Multiple ultrasound responses can induce repeated, slow rupture of liposomes, thus controlling the rate of drug delivery. Therefore, this drug delivery system contains a dual drug sustained-release delivery matrix. Furthermore, in evaluating related therapeutic effects, it was found that when the drug is KGN, it can simultaneously activate the ROS-induced mTOR signaling pathway, and the released KGN activates the SMAD5 signaling pathway. Both work synergistically to promote cartilage regeneration. Therefore, the ROS-responsive liposome system and the in-situ gel formation system work together to achieve the functions of drug sustained release and tissue regeneration. Under ultrasound control, the liposome rupture and drug release rate can be controlled, enabling better local drug delivery, multiple administrations, reduced patient discomfort, improved compliance and efficacy. This represents a new direction and product type in the field of tissue regeneration and drug delivery, suitable for industrial production.

[0034] In this invention, the preparation method has the following advantages: 1. By integrating multiple technologies, a sustained-release system with both sustained release and ultrasonic control is provided.

[0035] 2. Unlike conventional in-situ gels and thermosensitive gels, this invention can be controlled by ultrasound. By using sound control, the flow and formation within the in-situ composite hydrogel can be manipulated. The addition of nanoliposome technology reduces the degradation rate of the formed hydrogel, which is itself a major technological breakthrough in this field and opens up new directions for the use of in-situ hydrogels.

[0036] 3. It can provide a system that combines ROS and lipid-soluble drugs, simultaneously activating two mechanisms in vivo to achieve a synergistic effect and better exert the drug's efficacy. In particular, the discovery of KGN in the cartilage repair process was an unexpected technical effect we discovered when developing this mechanism. Attached Figure Description

[0037] Figure 1 Synthesis and characterization of precursors required for the preparation of reactive oxygen species (ROS) responsive liposomes; (ac) synthesis and... 1 1H NMR characteristic peak; (d) 1H NMR characteristic peak after TK fracture under H2O2 conditions.

[0038] Figure 2 The preparation process of ROS-responsive liposomes; Figure 3 Macroscopic images before and after gelation of in-situ nanocomposite hydrogels for controlled release of drugs in response to reactive oxygen species (ROS). Figure 4 KGN drug release curves under different ultrasound cycles; Figure 5 Biocompatibility of in-situ nanocomposite hydrogels for controlled release of reactive oxygen species (ROS)-responsive drugs. (a) Cytotoxicity at different ultrasound durations, (b) BMSC proliferation in the in-situ nanocomposite hydrogel, (c) Live / dead cell staining, (d) Semi-quantitative analysis of BMSC activity, (e) Determination of ROS generation and ROS scavenging by the DCFH-DA probe, (f) Semi-quantitative analysis of ROS generation.

[0039] Figure 6 The expression of chondrogenic genes after 7 days of culture; Figure 7 The expression of chondrogenic genes after 14 days of culture; Figure 8 The expression of chondroitin after 14 days of culture; Figure 9 Reactive oxygen species (ROS) responsive drug controlled-release in situ nanocomposite hydrogels promote chondrogenic differentiation by activating mTOR and Smad5 signaling. (a, c) Expression of mTOR and phosphorylated p-mTOR proteins, (b, d) Expression of Smad5 and phosphorylated p-Smad5 proteins, (e) Schematic diagram of the chondrogenic signaling pathway. Detailed Implementation

[0040] The following examples are used to further illustrate, but are not limited to, the present invention. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. The rats and mice described below were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0041] The beneficial effects of the traditional Chinese medicine composition described in this invention will be further illustrated through the following experiments.

[0042] Example 1 (1) Synthesis of reactive oxygen species (ROS) responsive distearylphosphatidylethanolamine-ketothiol-methoxy-polyethylene glycol (DSPE-TK-mPEG) precursor ① Synthesis of ketethiocarbamate (TK): Mercaptopropionic acid (11.44 g, 108.02 mmol), acetone (2.90 g, 49.1 mmol), and excess trifluoroacetic acid (TFA) were reacted at room temperature for 6 hours. The reaction product was then cooled in an ice bath until crystallization was complete. The solution was filtered and washed three times alternately with pre-cooled n-hexane and deionized water, and dried in a vacuum freeze-drying oven to obtain a white product.

[0043] ② Synthesis of DSPE-TK-mPEG: Ketothiolide (TK) (75.6 mg, 0.3 mmol), methoxyPEG-amino (150 mg, 0.03 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (230 mg, 1.2 mmol), and N-hydroxysuccinimide (NHS) (138 mg, 1.2 mmol) were completely dissolved in pH 6.8 phosphate buffer and stirred at room temperature for 3 days. To remove unreacted impurities, the reaction mixture was purified by dialyzing with deionized water at room temperature for 3 days using a dialysis bag (molecular weight cutoff [MWCO] = 1 kDa). The mixture was filtered through a 0.8 μm filter and then freeze-dried to obtain TK-mPEG. Subsequently, distearate phosphatidylethanolamine (DSPE) (12 mg, 0.016 mmol) was dissolved in 2 ml of chloroform and stirred at 60°C for 1 hour. TK-mPEG (54 mg, 0.024 mmol) and DCC (9.9 mg, 0.048 mmol) were dissolved in 2 ml of chloroform, and NHS (5.5 mg, 0.048 mmol) was dissolved in 1 ml of methanol. DCC and NHS were then added dropwise to the TK-mPEG solution. Subsequently, completely dissolved and activated DSPE was added dropwise to the mixture and reacted at room temperature for 3 days. The reaction solvent was evaporated using nitrogen, and the residue was dispersed with 5 ml of Tris-HCl buffer (pH 7.4). A mixture of 20 ml methanol and deionized water (1:1 v / v) was added, and the mixture was reacted at room temperature for 24 hours. The solution was then dialyzed for 24 hours using a dialysis bag (MWCO = 1 kDa), followed by dialyzed for another 24 hours using a dialysis bag with a molecular weight cutoff of 7 kDa. The resulting solution was filtered through a 0.8 μm filter and lyophilized for two days.

[0044] (2) Preparation of reactive oxygen species (ROS) responsive nanoliposomes ① Characterization of liposome raw materials: The synthesized compounds TK, TK-mPEG, and DSPE-TK-mPEG were dissolved in deuterated chloroform (5 mg / ml) and characterized using protons ( 1H) Nuclear magnetic resonance (NMR) was used to verify its chemical structure. Using H₂O₂ as an oxidant to simulate reactive oxygen species (ROS), TK (5 mg / ml) and hydrogen peroxide (100 mM) were co-incubated in the dark for 2 hours at a 1:1 (v / v) ratio. The product was then lyophilized and dissolved in deuterated water, and the chemical structure of the reaction product was determined by NMR.

[0045] ② Preparation of Liposomes@PpIX-KGN-Thrombin (Liposomes@PPIX-KGN-Thrombin): Liposomes@PPIX-KGN-Thrombin nanoparticles were prepared using a thin-film hydration and extrusion method. Hydrogenated soybean phosphatidylcholine (HSPC) and DSPE-TK-mPEG were dissolved in chloroform (6 ml) at a molar ratio of 95:5. Then, the sonosensitive agent (PpIX) and kartogenin (KGN) were dissolved in dimethylformamide (DMMF) and dimethyl sulfoxide (DMSO), respectively, and added to the above solution at 0.4% of the total liposome mass. The solution was evaporated at 50 °C using a rotary evaporator for 30 minutes to obtain a phospholipid membrane. Then, 6 ml of ultrapure water was added to the flask to fully dissolve the membrane. After dissolution, the membrane was hydrated at 65 °C for 1 hour. After cooling the mixed solution to 25 °C, 1000 UI of thrombin was added dropwise, and the mixture was vigorously stirred in an ice bath for 30 minutes. Finally, the resulting solution was extruded 30 times using a polycarbonate membrane (200 nm) to adjust its size. Then, it was dialyzed through a dialysis membrane (MWCO = 100 kDa) at 4°C in the dark for 24 h (with pre-cooled DI water replaced every 6 h) to remove free HSPC, DSPE-TK-PEG, PpIX, and KGN. The resulting nanoparticles were stored at -80°C. Furthermore, unmodified KGN liposomes@PpIX-thrombin (LPT), unmodified thrombin liposomes@PpIX-KGN (LPK), and liposomes@PpIX (LP) were synthesized using the same synthetic steps for further investigation.

[0046] The preparation method described above is described in [link to preparation instructions]. Figure 1 .

[0047] (1) Reactive oxygen species (ROS) responsive in-situ nanocomposite hydrogel gelation experiment 100 mg of fibrinogen was added to 1 ml of DPBS and dissolved in a 37°C water bath for 2 h until completely dissolved. The thrombin concentration (100 UI / ml) in the liposomes@PPIX-KGN-thrombin nanoparticles was quantified using the BCA method. Fibrinogen, the fibrin hydrogel precursor, was completely mixed with the liposomes@PPIX-KGN-thrombin nanoparticles at a 1:1 volume ratio. Then, the mixture was heated with a power of 1 W / cm².2 The mixed solution was sonicated for 3 minutes using an ultrasonic instrument to induce the formation of an in-situ hydrogel. See Figure 2 .

[0048] (2) Drug controlled release assay of in-situ nanocomposite hydrogels in response to reactive oxygen species (ROS) To evaluate the release behavior of KGN in fibrin-based smart hydrogels, this study used a fibrin-based smart hydrogel (liposomes@PPIX-KGN-thrombin). 100 mg / ml fibrinogen and 100 UI / ml thrombin were mixed at a 1:1 (v:v) ratio and sonicated to form a hydrogel. The fibrin hydrogel system was then placed in a dialysis bag (MWCO = 3500 Da) to test the KGN release behavior. Samples were subjected to KGN release at 1 W / cm² at 0, 1, 3, 5, and 11 days. 2 The hydrogel system was sonicated at a duty cycle of 50% (3 minutes). Samples were then immersed in 50 ml centrifuge tubes and rotated at 100 rpm in a constant-temperature shaker (37°C) to mimic human motion. 200 μL of solution was collected from the centrifuge tubes at the desired time points, and then the same volume of deionized water was added to the medium. The KGN concentration at each time point was determined by high-performance liquid chromatography (HPLC). See [link to relevant documentation]. Figure 3 .

[0049] (5) Physicochemical characterization of in-situ nanocomposite hydrogels responsive to reactive oxygen species (ROS) ① Scanning electron microscopy characterization: at a duty cycle of 50% and a power of 1 W / cm² 2 Under ultrasound conditions for 3 minutes, fibrin and thrombin-based hydrogel systems were formed, including fibrin hydrogel + ultrasound, fibrin hydrogel-liposome@PPIX-thrombin + ultrasound, and fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound hydrogel systems. These hydrogels were then freeze-dried. A conductive gold coating was then applied to the sample surface using a high-vacuum ion sputtering system. The surface morphology of the fibrin hydrogel + ultrasound, fibrin hydrogel-liposome@PPIX-thrombin + ultrasound, and fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound hydrogel scaffolds was observed using a field emission scanning electron microscope (SEM, JMF-7500F, Japan).

[0050] ② Mechanical Properties: The mechanical properties of hydrogels play a role in maintaining morphology and providing necessary mechanical support during tissue repair. This fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound in situ hydrogel formed cylindrical hydrogels with a diameter of 5 mm and a thickness of approximately 3 mm under different ultrasound durations. The mechanical properties of the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound nanoparticle in situ hydrogel were tested using an Anton Paar multi-functional indentation tester (Physica MCR301, MCR500).

[0051] ③ Swelling and Degradation Properties: The swelling and degradation rates of the fibrin hydrogel-liposome@PPIX-KGN-thrombin + sonicated in-situ nanohydrogel were evaluated using a gravimetric method. The dried sample (W0) was added to DPBS (5 ml) and placed in a constant-temperature shaker (Bluepard, sonicated, 37°C) at 100 rpm for 96 hours to swell. At the desired time points, the swollen hydrogel (W0) was weighed after removing the water. s Using equation E) s =[ (W s The expansion rate was estimated using the formula: (W0) / W0× 100% ] (n = 4). Furthermore, the degradation rate of the in-situ nanohydrogel was evaluated at the time points of interest using the same method.

[0052] Results and Discussion (1) Identification of the chemical structure of reactive oxygen species (ROS) responsive liposome precursors To verify the successful synthesis of TK with reactive oxygen species (ROS) sensitivity, the synthesized TK was dissolved in deuterated chloroform (CDCl3) and then... 1 Identification by ¹H NMR (AVANCE, 500 MHz). Spectral analysis yielded the following results: 1 ¹H-NMR: 2.85 (t, 4H), 2.58 (t, 4H), 1.58 (s, 6H), with chemical shifts and integrated areas corresponding to the characteristic peaks of TK, confirming the successful preparation of TK. Figure 1 a).

[0053] Furthermore, TK is embedded as an intermediate linker into mPEG and DSPE. Under the action of catalysts 1-C3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiophosphoramide (NHS), the carboxyl group of TK and the amino group of PEG are coupled, and the chemical structure of TK-mPEG is formed in CDCl3 via... 1 Identification by H NMR ( Figure 1(b) The results show that the characteristic peak at 1.52 ppm is the -CH3 group ("d" in the TK chemical structure), the characteristic peak (-CH2) is located at 2.729 ppm ("c" in the TK chemical structure), and the skeleton peak of mPEG (-CH2-CH2-) is located at 3.506 ppm ("a" in the TK-mPEG chemical structure), indicating that TK is successfully coupled to mPEG.

[0054] DSPE-TK-mPEG was synthesized by coupling TK-mPEG and DSPE via DCC / NHS. DSPE-TK-mPEG was synthesized in CDCl3 via... 1 HNMR confirmed ( Figure 1 c). The characteristic peak (-CO-) is located at 2.386 ppm, and there are also characteristic peaks of TK and mPEG, indicating that DSPE-TK-mPEG was successfully prepared by co-coupling TK-mPEG and DSPE through a two-step phthalamide condensation reaction.

[0055] To assess the sensitivity of TK to reactive oxygen species (ROS), TK was incubated with 100 μM H2O2 in the dark for 24 hours and then lyophilized. The product was then dissolved in deuterated water (D2O) and processed using... 1 H NMR assessment of the cleavage behavior of reactive oxygen species (ROS) on TK (TK) Figure 1 d). The results showed that a characteristic peak of thiol functional groups in the TK cleavage products was found at 2 ppm. This indicates that TK is reactive oxygen species (ROS) responsive, allowing for controlled TK cleavage.

[0056] (2) Preparation and characterization of reactive oxygen species (ROS) responsive nanoliposomes Liposome nanoparticles, as bioactive delivery carriers, have been widely used in controlled delivery systems and tissue regeneration. The successful preparation of responsive liposomes encapsulating drugs and thrombin is the most crucial step in this study. Therefore, this paper comprehensively evaluates the successful preparation of liposome@PpIX-KGN-thrombin nanocarriers using multiple characterization strategies. In this work, a reactive oxygen species (ROS)-responsive liposome was prepared via a thin-film hydration method, as described below. Figure 2 .

[0057] ① UV-Vis absorption spectrum: To evaluate the successful preparation of the liposome@PpIX-KGN-thrombin nanocarrier, qualitative analysis was performed using a UV-Vis spectrophotometer in the range of 240-600 nm. Absorption peaks for KGN and PpIX were detected at 274 nm and 410 nm, respectively, in the liposome@PPIX-KGN-thrombin aqueous solution. This indicates that the chondrogenic drug KGN and the sonosensitive agent PpIX were successfully loaded into the liposomes.

[0058] (3) Formation of in-situ nanocomposite hydrogels in response to reactive oxygen species (ROS) The preparation of the in-situ nanocomposite hydrogel is the core of this study. This in-situ hydrogel is formed in response to ultrasonic stimulation. Fibrinogen was dissolved at a concentration of 100 mg / ml in physiological saline at 37°C and incubated in a 37°C water bath for 1 hour to prepare a precursor solution. Furthermore, the thrombin concentration in the liposome@PPIX-KGN-thrombin nanocarrier was quantified using a BCA protein assay kit (Beyotime) and adjusted to 100 UI / ml. Then, the two precursors, fibrin hydrogel and liposome@PPIX-KGN-thrombin hydrogel, were thoroughly mixed at a 1:1 (V / V) ratio, and the same volume of precursor solution was delivered using a syringe. Under ultrasonic stimulation, the sonication agent (PpIX) generates reactive oxygen species (ROS). 1 O). Reactive oxygen species (ROS) trigger and induce the cleavage of TK (DSPE-TK-mPEG) in the phospholipid bilayer of the liposome@PPIX-KGN-thrombin nanocarrier, thereby causing the liposome@PPIX-KGN-thrombin nanocarrier to rupture. Thrombin and KGN are released from the liposome@PPIX-KGN-thrombin carrier. As described above, fibrinogen and thrombin contact to form a fibrinogen / liposome@PPIX-KGN-thrombin nanoparticle hydrogel. The precursor solution of the liposome@PPIX-KGN-thrombin hydrogel is liquid before exposure to ultrasound. However, the precursor solution is soluble in liquid after 3 minutes of ultrasound treatment (1 W / cm²). 2 A liquid-solid phase transition occurs rapidly afterward. Figure 3 This indicates that thrombin is released from liposomes and induces in situ hydrogelation.

[0059] (4) Swelling and degradation characteristics of in-situ nanocomposite hydrogels in response to reactive oxygen species (ROS) Furthermore, the degradation characteristics of biomaterials play a crucial role in practical applications, not only providing space for tissue regeneration but also regulating the tissue and cellular microenvironment. To simulate the joint microenvironment, three types of hydrogels were prepared and placed in centrifuge tubes containing 5 ml of DPBS, then incubated at 37°C on a shaker at 100 rpm. After incubation at predetermined time points, the hydrogels were removed and freeze-dried, and the remaining weights of the fibrin hydrogel, fibrin hydrogel-liposome@PPIX-KGN-thrombin, and fibrin hydrogel-liposome@PPIX-thrombin hydrogel were monitored. The results showed that the pure fibrin hydrogel degraded the fastest, decreasing to 28% of its original mass after 14 days. The remaining masses of the fibrin hydrogel-liposome@PPIX-KGN-thrombin and fibrin hydrogel-liposome@PPIX-thrombin hydrogels after 28 days in DPBS were 43.44% and 42.47% of their original masses, respectively. The results showed that the in-situ nanoparticle hydrogel exhibited superior degradation characteristics compared to the pure fibrin hydrogel. This may be because the nanoparticles enhanced the cross-linking ability of fibrinogen and thrombin, thus delaying the degradation of the hydrogel to some extent, which would be more conducive to the sustained release of drugs in the hydrogel.

[0060] (5) Mechanical properties of reactive oxygen species (ROS) responsive in-situ nanocomposite hydrogels To investigate the mechanical properties of ultrasound-responsive reactive oxygen species (ROS) hydrogels, the precursor solution was sonicated for 1–5 minutes. After in-situ hydrogel formation, the mechanical properties of the liposome@PPIX-KGN-thrombin hydrogel were measured using a nanoindenter. The results showed that the compressive modulus of the hydrogel gradually increased with increasing sonication duration. However, when the sonication time exceeded 3 minutes, the compressive modulus only increased slightly, possibly because the hydrogelation of the precursor solution prevented thrombin from fully participating in cross-linking. Furthermore, compared to pure fibrin hydrogel, the compressive modulus of the fibrin hydrogel-liposome@PPIX-KGN-thrombin hydrogel increased by 47.89% after 3 minutes of sonication. This indicates that the presence of liposomes effectively improves the mechanical stress of the hydrogel.

[0061] (6) Analysis of drug release behavior of in-situ nanocomposite hydrogels in response to reactive oxygen species (ROS) Encapsulation efficiency and release characteristics of drugs play a crucial role in drug delivery systems (DDS). Lipid-soluble KGN and PpIX are readily loaded into the phospholipid bilayer of liposomes, achieving high encapsulation efficiencies. High-performance liquid chromatography (HPLC) determined the encapsulation efficiencies of KGN and PpIX in liposomes@PPIX-KGN-thrombin to be 84.2% and 86.7%, respectively. Furthermore, the encapsulation efficiency of PpIX in LPT was 89.3%. The similar encapsulation efficiencies of KGN and PpIX loaded in liposomes@PPIX-KGN-thrombin and LPT may be due to the lower drug-liposome ratio (0.4%), which did not reach the maximum loading value of the liposomes. To investigate the ultrasound-induced release performance of KGN, fibrin hydrogel-liposomes@PPIX-KGN-thrombin + ultrasound hydrogel (KGN, 95.2 μg / ml) was treated with and without continuous ultrasound (days 0, 1, 3, 5, and 11) at 1 W / cm². 2 The release rate of KGN was assessed by sonication (3 minutes). Fibrin hydrogel-liposome@PPIX-KGN-thrombin hydrogel was placed in a dialysis bag (10000-14000 DW) and then in a centrifuge tube containing 7.5 ml DPBS. The mixture was shaken at 120 rpm at 37°C, and 200 μL was collected at different time points for detection. The results showed that the release efficiency of KGN gradually increased with the number of sonication treatments. However, when the sonication frequency exceeded 3 times, the release rate reached a threshold (86%). Figure 4 To further investigate the regularity of KGN release, the cumulative KGN release from fibrin hydrogel-liposome@PPIX-KGN-thrombin nanogel (100 μg) was measured after sonication. The results showed that KGN was gradually released after sonication, reaching the release threshold after 12 days. Importantly, the release rate after three sonications (85.66%) was significantly higher than that after only one sonication (42.33%), which may be because more KGN was released from liposome@PPIX-KGN-thrombin through prolonged sonication. To achieve good therapeutic effects, a multiple sonication strategy was employed according to the above time points during long-term in vitro cell culture and in vivo treatment.

[0062] Example 2 (1) Cell source and culture method All BMSCs discussed in this section were derived from the femoral bone marrow of SD rats. Rats were euthanized after anesthesia with sodium pentobarbital and sterilized by immersion in 75% anhydrous ethanol. The femur was removed, and the femoral medullary cavity was flushed with 2% penicillin-dextrose antimicrobial agents. After filtration, the extract collected from the femoral medullary cavity was cultured in a complete medium containing α-MEM, 1% PS, and 10% FBS. Primary BMSCs were purified in passages 3-4 for further studies.

[0063] (2) 3D cell culture using reactive oxygen species (ROS) responsive in-situ nanocomposite hydrogels To evaluate the biological properties of the hydrogel system in vitro, passaged BMSCs were used at a density of 1 × 10⁶ cells per hydrogel scaffold. 5 Cells were seeded into a 3D in situ hydrogel. Specifically, BMSCs were resuspended in a 100 mg / ml fibrinogen solution and the density was adjusted to 2 × 10⁻⁶ cells / mL. 7 Cells / ml. Various types of NPs (100 UI / ml, thrombin) and fibrinogen were added to a cylindrical mold (Ф4 mm × 2 mm) at a ratio of 1:1 (20 μl). The hydrogel precursor solution was added at 1 W / cm². 2 The cells were sonicated at 50% power and 50% duty cycle for 3 minutes to form hydrogels in the dark. Furthermore, the smart in-situ hydrogels were grouped as follows: (1) fibrin hydrogel (FT); (2) fibrin hydrogel + ultrasound (FT+US); (3) fibrin hydrogel-liposome@PPIX-thrombin + ultrasound (FLPT+US); and (4) fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound (FLPKT+US). After gelation, the cell scaffold constructs were cultured in complete culture medium for further studies. The fibrinogen precursor was sterilized by UV treatment for one hour and then used in vitro and in vivo.

[0064] In the attached figures, the order of the bars (FT), (FT+US), (FLPT+US), and (FLPKT+US) in 5, 6, 7, and 9 is arranged from left to right.

[0065] (3) Cell biological compatibility experiment of in-situ nanocomposite hydrogels responding to reactive oxygen species (ROS). This study used a CCK-8 assay kit to detect cell viability in fibrin-based hydrogels. The cell hydrogel structures were placed in 96-well plates and incubated with 150 μL of complete culture medium for 24 hours, after which the medium was replaced with fresh medium containing 10% CCK-8 reagent. After incubation in a cell culture incubator for 1.5 hours, the absorbance of the supernatant was analyzed at 450 nm using a multi-mode microplate reader, and cell viability was calculated. To assess BMSC proliferation within the hydrogel, cells were cultured in the hydrogel for 1, 3, and 7 days using the same measurement method described above.

[0066] (4) AM / PI staining The activity of BMSCs loaded in fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound nanogel was visualized using a live / dead cell staining kit. After 3D culture for 1, 3, and 7 days, BMSCs (1×10⁻⁶ cells / ... 5 The samples were then labeled for liveness and inactivation. The samples were then washed three times with DPBS, and the 3D proliferation status of BMSCs was analyzed using a CLSM (FV30-ILSW, Olympus, Japan) with emission at 494 nm and excitation at 528 nm.

[0067] (5) Detection of intracellular reactive oxygen species (ROS) To detect intracellular reactive oxygen species (ROS) in vitro, the ROS probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Dojin Chemicals, Japan) was used in fibrin hydrogels, fibrin hydrogels with sonication, fibrin hydrogel-liposomes@PPIX-thrombin+sonication, and fibrin hydrogel-liposomes@PPIX-KGN-thrombin+sonication hydrogels to test ROS in BMSCs in vitro. The hydrogel precursor was mixed with BMSCs (5 × 10⁻⁶ cells / year). 5 The DCFH-DA probe (10 mM) was mixed and preloaded. Four hours after sonication, intracellular fluorescence intensity was analyzed and photographed using CLSM. Additionally, the reactive oxygen species (ROS) scavenger N-acetylcysteine ​​(NAC, 50 mM) was added to the culture medium to inhibit ROS production, serving as a control group (see 5e-f).

[0068] (6) In vitro chondrogenicity-promoting experiment of in-situ nanocomposite hydrogels in response to reactive oxygen species (ROS). ① Quantitative real-time polymerase chain reaction (qRT-PCR) analysis: After 3D culture of BMSCs in a smart in-situ hydrogel, gene expression levels at different time points were analyzed by qRT-PCR. BMSCs (5 × 10⁻⁶) were cultured in this manner. 7The fibrinogen precursor was resuspended in 1 ml of fibrinogen precursor (100 mg / ml), and an equal volume of liposome@PPIX-KGN-thrombin nanoparticle solution (thrombin, 100 UI / ml) was added. Then, 100 μl of the hydrogel precursor was sonicated for 3 minutes to gel. After incubation in 6-well plates for 24 hours, the medium was replaced with chondrogenic medium containing high-glucose DMEM, 10% FBS, 1% PS, 50 μg / ml ascorbic acid, 100 mM dexamethasone, and 1% ITS premix for 7 and 14 days. The fibrin hydrogel + sonication, fibrin hydrogel-liposome@PPIX-thrombin + sonication, and fibrin hydrogel-liposome@PPIX-KGN-thrombin + sonication hydrogel groups were sonicated on days 0, 3, 5, and 11. The cultured hydrogel structures were frozen in liquid nitrogen and homogenized for 30 seconds, followed by mRNA extraction using an RNAiso kit (TaKaRa, Japan). A reverse transcription kit (TaKaRa, Japan) was used for reverse transcription (RT) to obtain cDNA. qRT-PCR was performed using a TB Green qPCR (TaKaRa, Japan) detection system. The qRT-PCR cycling conditions were 95 °C for 3 min, followed by 95 °C for 3 s, 60 °C for 30 s, for a total of 42 cycles.

[0069] (7) Evaluation of the effect of hydrogel on cartilage repair in animals ① Macroscopic observation: Articular cartilage from the femoral condyles of SD rats was obtained at 4 and 8 weeks for macroscopic observation. In addition, the morphological effects of the cartilage treatment were evaluated according to the O'Driscoll scoring system in each group.

[0070] ② Atomic Force Microscopy (AFM) Analysis: To assess the effect of surface roughness on cartilage defect regeneration, AFM was used to evaluate the cartilage regeneration area in rats. AFM was performed on all samples (n=4) using a Nanoscope V Multimode 8 scanning probe. All experiments were conducted using the same AFM probe at 25°C and 25% relative humidity.

[0071] ③ Histological and Immunohistochemical Analysis: Femurs from SD rats were collected and fixed in 4% formalin for 2 days. The femoral condyles of the SD rats were then decalcified in a decalcification solution for 4 weeks. After graded ethanol dehydration, the femurs were paraffin-embedded in suitable locations and sectioned into 10 mm sections using a microtome. Femoral sections from different groups were stained with Red & Green and hematoxylin-eosin (H&E). The expression of collagen I and collagen II was then analyzed by immunohistochemical staining of the defect areas. Staining was performed with DAB solution and counterstained with hematoxylin. Staining images were obtained using an inverted microscope. Furthermore, at 8 weeks, the cellular status of major organs (heart, liver, spleen, lung, and kidney) was examined by pathological staining (H&E) to assess the long-term safety of the drug formulation.

[0072] (8) Biocompatibility of Reactive Oxygen Spectroscopy (ROS)-Response In-situ Nanocomposite Hydrogels The biocompatibility of hydrogels plays a crucial role in maintaining cell viability, proliferation, and differentiation in tissue engineering. In this work, cell viability in fibrin hydrogel-liposome@PPIX-KGN-thrombin nanocomposite hydrogels was assessed using a CCK-8 assay. BMSCs were seeded in various hydrogels for 24 hours with or without exposure to sonication (3 minutes). There was no difference in cell viability between the fibrin hydrogel group and the fibrin hydrogel + sonication group, indicating that 3 minutes of sonication application had no effect on cells.

[0073] Fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound and fibrin hydrogel-liposome@PPIX-thrombin + ultrasound in situ nanocomposite hydrogels did not exhibit significant toxicity compared to pure fibrin hydrogels, indicating excellent biocompatibility. The results showed that the residual reactive oxygen species (ROS) after the redox reaction with liposomes (TK) (generated by ultrasound, 3 min) did not have additional toxic side effects on BMSCs. To further explore the intrinsic relationship between cell viability in the hydrogels and ultrasound duration, four types of hydrogels were exposed to ultrasound for different durations (1–5 min). In all groups, there was no statistically significant difference in cell viability within three minutes of ultrasound exposure. However, mild toxicity was observed when the ultrasound duration exceeded four minutes. Furthermore, cell viability gradually decreased with increasing ultrasound duration, suggesting that the increase in reactive oxygen species (ROS) with increasing ultrasound time can damage cells. Figure 5 a).

[0074] The proliferation rate of BMSCs in the four groups of 3D hydrogels at 1, 3, and 7 days was quantitatively analyzed using the CCK-8 assay. OD values ​​represent the number of cells in each hydrogel group. The results showed that the OD value gradually increased with culture time, indicating a gradual increase in the number of BMSCs in the hydrogels. Furthermore, the OD value of the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group was higher than that of the fibrin hydrogel group, indicating that the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group was more conducive to cell proliferation (5b). Visual analysis was performed using a live / dead cell assay. Figure 5 c) Live cells showed green fluorescence staining, while dead cells appeared red. The results indicated that almost no red fluorescence was observed in the fibrin hydrogel-liposome@PPIX-KGN-thrombin + sonication in situ hydrogel from day 1 to day 7, demonstrating excellent biocompatibility. Furthermore, semi-quantitative analysis of live / dead staining using ImageJ showed that over 90% of BMSCs were detected in both the fibrin hydrogel-liposome@PPIX-thrombin + sonication and fibrin hydrogel-liposome@PPIX-KGN-thrombin + sonication in situ hydrogels. Figure 5 d). Therefore, the in-situ nanocomposite hydrogel demonstrates good biocompatibility. Furthermore, the in-situ nanocomposite hydrogel promotes the migration and proliferation of encapsulated BMSCs.

[0075] (9) The ability of reactive oxygen species (ROS) to promote chondrogenesis in situ nanocomposite hydrogels in vitro This article further analyzed the effects of in situ nanocomposite hydrogels on the differentiation of rat BMSCs into cartilage using Alcian blue staining, qRT-PCR, and Western blotting (WB).

[0076] ① Alcian Blue Staining: The ECM of cartilage, including proteoglycans and collagen II, can be detected by Alcian blue staining. Alcian blue staining showed that after BMSCs were co-cultured with each group of hydrogels for 7 days, proteoglycan expression (blue) gradually increased in the fibrin hydrogel, fibrin hydrogel + ultrasound, fibrin hydrogel-liposome@PPIX-thrombin + ultrasound, and fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound groups. The results indicate that the fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound group has a significant ability to induce cartilage differentiation. In addition, the proteoglycan expression (blue) in the fibrin hydrogel-liposome@PPIX-thrombin + ultrasound group was significantly different from that in the fibrin hydrogel group, indicating that an appropriate amount of reactive oxygen species (ROS) can promote cartilage regeneration.

[0077] ② Expression of qRT-PCR-related cartilage genes: To further analyze cartilage differentiation in BMSCs, the expression of cartilage-specific markers (such as Sox9, ACAN, and Col II) was measured by real-time PCR. The fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group showed higher expression of cartilage-related genes at different time points than other groups. Compared with the fibrin hydrogel group, the levels of Sox9, ACAN, and Col II genes in the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group increased by 7.31, 8.11, and 10.49 times, respectively, on day 7. Compared with day 7, the expression levels on day 14 increased by 1.98, 2.29, and 2.83 times, respectively. Furthermore, the fibrin hydrogel-liposome@PPIX-thrombin+ultrasound group also showed significantly higher expression levels of cartilage-related genes on days 7 and 14 compared to the fibrin hydrogel group, suggesting that reactive oxygen species (ROS) may enhance cartilage regeneration capacity. Figure 6-7 ).

[0078] ③ Expression of Western blot-related chondroitin proteins: The expression of cartilage-specific markers (such as Sox9, ACAN, and ColII) was measured by Western blot. Consistent with the results of real-time quantitative PCR, the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group also showed increased protein expression of ACAN, Sox9, and ColII. Figure 8 ).

[0079] (10) Mechanism analysis of reactive oxygen species (ROS) response in situ nanocomposite hydrogel to promote cartilage regeneration ① In-situ nanocomposite hydrogels modulate the generation of reactive oxygen species (ROS) and activate the mTOR signaling pathway: mTOR signaling is an important signaling pathway in the generation of reactive oxygen species (ROS). To further investigate the regulation of the mTOR signaling pathway by reactive oxygen species (ROS), the expression levels of p-mTOR (phosphorylation) in different groups were analyzed. For example... Figure 9As shown in a and 9c, ultrasound induces the production of reactive oxygen species (ROS). Western blotting results showed that the p-mTOR levels in the fibrin hydrogel-liposome@PPIX-thrombin+ultrasound group and the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group were increased, while the mTOR phosphorylation level in the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group was significantly reduced by 84.6% and 103.5%, respectively. After culture with NAC (a reactive oxygen species (ROS) scavenger), the mTOR phosphorylation level in the fibrin hydrogel-liposome@PPIX-thrombin+ultrasound and fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound groups was significantly reduced.

[0080] ② Reactive Oxygen Species (ROS) Response in In-situ Nanocomposite Hydrogel Activation of the Smad5 Signaling Pathway: Kartogenin (KGN) is an activator of the Smad4 / Smad5 signaling pathway. Released from liposomes, it promotes chondrocyte differentiation. After 14 days of culture in the hydrogel, the level of phosphorylated Smad5 in BMSCs was analyzed, and it increased by 179.12% in the fibrin hydrogel-liposome@PPIX-KGN-thrombin+ultrasound group. Figure 9 (b, d). Schematic diagram of the mTOR / Smad5 signaling pathway activated by ultrasound and KGN is shown below. Figure 9 As shown in e.

[0081] in conclusion: The bioactivity and biocompatibility of fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel were evaluated at the cellular level, demonstrating its high biocompatibility. Using rat-derived bone marrow mesenchymal stem cells (BMSCs) as the experimental cell line, migration and differentiation experiments of the fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel were conducted. The results showed that the fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel group promoted cell migration and enhanced BMSC differentiation into cartilage. The fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel group promoted cartilage regeneration by regulating the mTOR and SMAD5 signaling pathways. Finally, in a rat model of cartilage defects, the fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel group significantly improved cartilage repair.

[0082] (1) A three-step method was used to synthesize DSPE-TK-mPEG, and the successful preparation of the material was confirmed by 1H NMR spectroscopy. After TK was co-cultured with H2O2 at 37°C in the dark, a characteristic peak of the thiol functional group in the TK cleavage product was detected at 2 ppm. This indicates that the synthesized liposome precursor has good reactive oxygen species (ROS) responsiveness, which is suitable for the subsequent synthesis of responsive nanoliposomes.

[0083] (2) Thrombin, the small molecule drug KGN, and the sonosensitive agent (PpIX) nanoliposomes were successfully prepared by thin-film hydration and extrusion methods. The lipid-soluble drugs KGN and PpIX exhibited high drug loading rates in the liposome bilayer. The PLKT nanoparticles were characterized by TEM, UV-vis, and DLS.

[0084] (3) Fibrin hydrogel-liposome@PPIX-KGN-thrombin precursor solution was used to form in-situ hydrogels at a 1:1 (V / V) ratio under different sonication times. Based on this, the swelling and degradation properties of the responsive nanohydrogel were analyzed. The results showed that the responsive in-situ nanohydrogel could effectively improve the cross-linking strength inside the hydrogel and prolong the degradation time of the hydrogel.

[0085] (4) The drug release and mechanical properties were analyzed, and a reasonable ultrasound time was optimized. The results showed that the mechanical properties gradually increased with increasing ultrasound time, and the mechanical stress threshold could be obtained after 3 minutes. The effect of different ultrasound times on the drug release behavior of fibrin hydrogel-liposome@PPIX-KGN-thrombin hydrogel was investigated. Satisfactory drug release effect was obtained by three cycles of ultrasound, each lasting 3 minutes. Under ultrasound stimulation, the drug release behavior was precisely regulated by ultrasound, demonstrating the ability to release drugs in an intelligent ultrasound response.

[0086] (5) The cytotoxicity of fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel was first evaluated. The potential toxicity of fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel to rat-derived mesenchymal cells was detected by the CCK-8 assay at different sonication times. The results showed that the fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel had no significant toxicity within 3 minutes of sonication. Therefore, the sonication time of fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel was controlled at 3 minutes, and the power was 1 W / cm². 2 .

[0087] (6) The activity and proliferation of BMSCs in different groups of hydrogels at 1, 3 and 7 days were evaluated by CCK-8 and Annexin-V-FITC / PI double staining experiments. The results showed that all groups of hydrogels had good cell activity, and the fibrin hydrogel-liposome@PPIX-KGN-thrombin nanohydrogel group was more conducive to BMSC proliferation.

[0088] (7) The fluorescence intensity of intracellular reactive oxygen species (ROS) in different hydrogels was detected using the DCFH-DA fluorescent probe to assess the amount of intracellular ROS produced after 3 minutes of sonication. The results showed that no significant ROS fluorescence was produced in the pure hydrogel group and the sonicated group; both sonicated nanohydrogels showed significant fluorescence, and there was no significant difference between them; when ROS inhibitors were added, the fluorescence intensity decreased significantly in both groups.

[0089] (8) This study used QRT-PCT, WB, and Alcian blue staining to verify the ability of BMSCs to differentiate into chondrocytes in vitro. The results showed that the chondrocyte differentiation ability of fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound was higher than that of the fibrin hydrogel + ultrasound and fibrin hydrogel-liposome@PPIX-thrombin + ultrasound groups. This indicates that fibrin hydrogel-liposome@PPIX-KGN-thrombin + ultrasound has good chondrocyte differentiation ability and can be used to realize the potential of cartilage regeneration. Furthermore, the differentiation ability of the fibrin hydrogel-liposome@PPIX-thrombin + ultrasound group was also slightly higher than that of the control group, indicating that an appropriate amount of reactive oxygen species (ROS) can promote cartilage regeneration.

[0090] (9) To further explain the deeper understanding of cartilage repair by the materials designed in this paper, the mTOR and SMAD5 signaling pathways were verified at the cellular level through WB experiments. This demonstrated that the reactive oxygen species (ROS) generated by fibrin hydrogel-liposome@PPIX-KGN-thrombin nanogel can activate the mTOR signaling pathway, and the released KGN can activate the SMAD5 signaling pathway. The two work together to promote cartilage regeneration.

Claims

1. A method for preparing an in-situ nanocomposite hydrogel with controlled drug release, characterized in that, Includes the following steps: 1) Synthesize the liposome precursor DSPE-TK-mPEG with reactive oxygen species response; 2) React the DSPE-TK-mPEG obtained in step 1) with liposome components to prepare nanoliposomes LP with reactive oxygen species response; the synthesis of DSPE-TK-mPEG adopts a three-step strategy, which is divided into three steps: ketithiothiol TK synthesis, TK-mPEG synthesis and DSPE-TK-mPEG synthesis. 3) Prepare nanoliposomes LPD by loading the lipid-soluble drug D onto LP in step 2). 4) Load thrombin into the LPD obtained in step 3) to obtain nanoliposomes LPDT; 5) Add the LPDT from step 4) to the sound-sensitizing agent, so that the sound-sensitizing agent is embedded inside the LPDT, to obtain LPDT containing the sound-sensitizing agent; 6) Mix the LPDT containing the acoustic sensitizer obtained in step 5) with fibrinogen to obtain an in-situ nanocomposite hydrogel precursor solution for controlled release of reactive oxygen species-responsive drugs. 7) Under ultrasound stimulation, the precursor solution obtained in step 6) forms an in-situ nanocomposite hydrogel for controlled release of the drug.

2. The preparation method according to claim 1, characterized in that, The synthesis steps of the ketethiothrin TK are as follows: after reacting mercaptopropionic acid, acetone, and trifluoroacetic acid, crystallize, filter, and wash, the ketethiothrin TK is obtained.

3. The preparation method according to claim 1, characterized in that, The TK-mPEG synthesis steps are as follows: Ketothiolide TK, methoxyPEG, aminom-PEG, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC are reacted at room temperature with stirring, dialyzed, and then filtered to obtain the TK-mPEG.

4. The preparation method according to claim 1, characterized in that, The synthesis steps of DSPE-TK-mPEG are as follows: TK-mPEG is dissolved to obtain a TK-mPEG solution, DCC and NHS are added dropwise to the TK-mPEG solution for reaction, then distearate phosphatidylethanolamine DSPE solution is added dropwise and reacted at room temperature, and the DSPE-TK-mPEG is obtained after dialysis.

5. The preparation method according to claim 1, characterized in that, The acoustic sensitizer is selected from one or more of HpD, PpIX, and Ce6.

6. The preparation method according to claim 1, characterized in that, The lipid-soluble drug D is selected from Kartogenin or doxorubicin.

7. The preparation method according to claim 1, characterized in that, The fibrinogen and thrombin in the liposomes were mixed in a volume ratio of 1:0.5-3, with concentrations of 20-400 mg / ml and 2-1000 UI / ml, respectively.

8. The preparation method according to claim 1, characterized in that... The ultrasonic power range is 0.5-5 W / cm². 2 The ultrasound time is 1-5 minutes.

9. The in-situ nanocomposite hydrogel with controlled drug release obtained by the preparation method of claim 1.

10. The in-situ nanocomposite hydrogel with controlled drug release according to claim 9, characterized in that, The lipid-soluble drug D is Kartogenin.

11. The application of the in-situ nanocomposite hydrogel with controlled drug release as described in claim 9 in the preparation of cartilage repair drugs.

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