Sequential drug release anti-adhesion nanocomposite hydrogel preparation and preparation method and application thereof

Abstract

This invention discloses a method for preparing a nanocomposite hydrogel and its application in preventing postoperative tissue adhesions. The hydrogel consists of a basic framework of adipate dihydrazide-functionalized polysaccharide-drug-loaded nanomicelles cross-linked via dynamic acylhydrazone bonds. Further, benzodiazepine is formed through a click reaction between a dihydrolipoic acid prodrug molecule containing 2-formylphenylboronic acid and the functionalized polysaccharide, reinforcing the cross-linking density of the gel network and endowing it with reactive oxygen species-responsive sequential drug release functionality. This hydrogel possesses strong mechanical strength and can adhere to the wound surface, effectively isolating damaged peritoneum. In the highly reactive oxygen species environment of the postoperative wound, the benzodiazepine bonds break, releasing dihydrolipoic acid, achieving a dual cascade scavenging of reactive oxygen species, thereby alleviating inflammatory responses and oxidative stress. Subsequently, the pore size of the gel network increases, accelerating the release of antifibrotic drugs loaded in the micelles, thus effectively inhibiting the TGF-β1-mediated fibrosis process. Ultimately, this achieves effective prevention and treatment of postoperative adhesions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a sequential drug-release nanocomposite hydrogel and its application in preventing postoperative tissue adhesion. Background Technology

[0002] Postoperative adhesions are a common complication following trauma, abdominal, or pelvic surgery, with an incidence rate exceeding 90%. They can lead to serious complications such as intestinal obstruction, chronic abdominal and pelvic pain, and female infertility, significantly reducing patients' quality of life and even endangering their lives. Clinically, adhesiolysis is commonly used to relieve postoperative adhesions; however, patients often experience recurrent adhesions, which are considered more complex and difficult to prevent than primary adhesions. Furthermore, secondary surgery and hospitalization significantly increase medical costs. Therefore, there is an urgent need to develop effective adhesion prevention strategies.

[0003] Existing techniques for preventing postoperative tissue adhesions mainly fall into three categories: improving surgical skills, applying medication locally or systemically, and implanting physical barriers between damaged tissue and adjacent organs. However, drug therapy has limitations due to issues such as rapid clearance and unavoidable toxic side effects. Solid film barriers (such as...) It has drawbacks such as not being able to perfectly fit irregular wounds, being prone to deformation during use, and being difficult to operate. Liquid barriers (such as...) Due to their high fluidity, hydrogels are difficult to retain for long periods on wound surfaces. In contrast, hydrogel barriers, with their superior coverage compared to solid films, superior retention compared to liquid barriers, good coverage of irregular wounds, and compatibility with minimally invasive surgery, have become the most commonly used and widely applied barrier type for preventing postoperative tissue adhesions. Furthermore, the inherent biocompatibility and biodegradability of hydrogels make them suitable as drug delivery platforms. However, existing commercially available hydrogel barriers still have drawbacks such as rapid degradation, poor mechanical properties, lack of pharmacological functions, and only functioning as a single physical barrier, making it difficult to prevent adhesion formation from the root cause of the disease.

[0004] Studies have shown that postoperative adhesion formation involves a series of complex pathological processes. Based on its development, it can be divided into several key stages: the coagulation and inflammation stage, the fibrinolytic stage, and the fibrosis and tissue remodeling stage. Among these, inflammatory response / oxidative stress and mesothelial-mesenchymal transition (MMT) of mesothelial cells are key pathological events in the inflammatory and fibrotic stages of postoperative adhesion formation. In recent years, some researchers have attempted to introduce reactive oxygen species scavenging components or anti-inflammatory molecules into barrier materials to alleviate inflammatory response / oxidative stress and thus enhance the anti-adhesion effect of the barrier materials. In addition, a very small number of studies have attempted to use the barrier materials themselves or encapsulate bioactive molecules within them to inhibit the MMT process of mesothelial cells, thereby reducing adhesion formation. However, a single therapeutic mechanism (e.g., anti-inflammatory, antioxidant, or MMT inhibition) is unlikely to reverse the complex pathological cascade of adhesion formation. Novel bio-barrier materials that can alleviate the inflammatory pathological microenvironment of the postoperative peritoneum and simultaneously inhibit mesothelial cell MMT may hold promise for fundamentally inhibiting the occurrence of postoperative adhesions, as they can act on key events throughout the entire adhesion formation process. However, designing hydrogel materials that can intelligently target and form different key stages of cascade adhesion in time and space, while also possessing excellent physical barrier properties, remains a huge challenge in clinical practice. Summary of the Invention

[0005] Therefore, the present invention aims to provide a sequential-release anti-adhesion nanocomposite hydrogel formulation and its preparation method. The hydrogel is based on a functionalized polysaccharide-drug-loaded nanomicelle composite system with dynamic acylhydrazone bond crosslinking. Benzodiazepines are further formed through a click chemistry reaction between PLA molecules containing 2-FPBA (dihydrolipoic acid prodrug) and the functionalized polysaccharide, reinforcing the crosslinking density of the gel network and endowing the gel with reactive oxygen species (ROS) responsive sequential drug release functionality. This hydrogel exhibits strong mechanical strength, rapid self-healing ability, and good tissue adhesion, effectively adhering to the wound surface to isolate damaged peritoneum. In the postoperative wound environment with high ROS, ROS cleave the benzodiazepine bonds to release the antioxidant drug dihydrolipoic acid, achieving a dual cascade scavenging of ROS, thereby alleviating excessive inflammatory response and oxidative stress. Simultaneously, with the destruction of crosslinking points in the gel network, the pore size of the gel network increases, accelerating the release of the antifibrotic drug loaded in the micelles, thereby effectively inhibiting the TGF-β1-mediated MMT process and alleviating the fibrosis process. In summary, this hydrogel effectively prevents and treats postoperative adhesions.

[0006] To achieve its purpose, the technical solution adopted by this invention is as follows:

[0007] A sequential-release drug-anti-adhesion nanocomposite hydrogel formulation, characterized in that the hydrogel formulation is constructed in the following manner:

[0008] a) A gel network basic structure is formed by cross-linking adipate dihydrazide-functionalized polysaccharide with drug-loaded nanomicelles via dynamic acylhydrazone bonds;

[0009] b) Introduce PLA (2-FPBA-SRS-FPBA-2, where SRS represents dihydrolipoic acid and 2-FPBA is 2-formylphenylboronic acid), a reactive oxygen species responsive dihydrolipoic acid prodrug, into the basic structure of the gel network. Benzodiazepine is formed through a click chemical reaction between 2-FPBA on the molecule and the acylhydrazide group derived on the polysaccharide chain, thereby strengthening the crosslinking density of the gel network and endowing the gel with reactive oxygen species responsive sequential drug release function.

[0010] The drug-loaded nanomicelles are composed of aldehyde-functionalized Pluronic F127 (PF127) encapsulating anti-fibrotic drugs; the sequential drug release occurs when the hydrogel is in a high-level reactive oxygen species environment, the reactive oxygen species cleave the benzodiazepine bond to release the antioxidant drug dihydrolipoic acid, and the pore size of the gel network increases accordingly, accelerating the release of the anti-fibrotic drug.

[0011] The polysaccharide is any one of hyaluronic acid, chondroitin sulfate, fucoidan, and sodium alginate; the antifibrotic drug is any one of pirfenidone, nintedanib, cryptotanshinone, tanshinone, curcumin, and baicalein.

[0012] The nanomicelles have a particle size distribution in the range of 0-50 nm, a drug encapsulation efficiency of ≥60%, and a drug loading of ≥0.01 wt%. The aldehyde functionalization degree of PF127 is 2 aldehyde groups / PF127 molecule. The degree of substitution of adipic acid dihydrazide in the adipic acid dihydrazide functionalized polysaccharide is 30-80%, and the molecular weight ranges from 10-500 kDa.

[0013] This invention also provides a method for preparing a sequential-release drug anti-adhesion nanocomposite hydrogel formulation, comprising the following steps;

[0014] Step 1: Synthesize adipic acid dihydrazide-functionalized polysaccharides via an amide condensation reaction between the carboxyl groups on the polysaccharide chain and adipic acid dihydrazide;

[0015] Step 2: Synthesize PF127 modified with aldehydes at both ends and prepare PF127 drug-loaded nanomicelles;

[0016] Step 3: Synthesize PLA, a reactive oxygen species-responsive dihydrolipoic acid prodrug molecule with 2-formylphenylboronic acid grafted with thiol groups at both ends;

[0017] Step 4: Mix PLA with PF127 drug-loaded nanomicelle solution to prepare mixed precursor solution, and mix the mixed precursor solution with adipic acid dihydrazide functionalized polysaccharide solution to obtain sequential release drug anti-adhesion nanocomposite hydrogel formulation.

[0018] In some embodiments, the concentration of the adipic acid dihydrazide-functionalized polysaccharide solution before mixing is 1-5 wt%, the concentration of the PF127 drug-loaded nanomicelle solution is 5-25 wt%, the concentration of the PLA solution is 0.1-0.5 wt%, the mixing mass concentration ratio of the adipic acid dihydrazide-functionalized polysaccharide solution to the PF127 nanomicelle solution is 8:1-1:4, and the gel precursor solution is mixed by pre-mixing or partial mixing.

[0019] Another object of the present invention is to provide the use of a sequential-release drug-resistant anti-adhesion nanocomposite hydrogel formulation in the preparation of a barrier material for preventing postoperative tissue adhesion.

[0020] In some embodiments, the postoperative tissue adhesion type is any one or more of abdominal adhesions and pelvic adhesions.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) A novel reactive oxygen species-responsive dihydrolipoic acid prodrug PLA containing 2-formylphenylboronic acid was designed and synthesized. It not only serves as a crosslinking monomer to construct a ROS-responsive gel network, but also scavenge reactive oxygen species through bond breaking and drug release cascade, thereby alleviating postoperative excessive inflammatory response and oxidative stress.

[0023] (2) Inspired by the high reactive oxygen species level in the postoperative wound, a PLA-based reactive oxygen species responsive nanocomposite hydrogel was designed. After responding to the reactive oxygen species level in the wound, it realizes the temporal and spatial controllable sequential release of anti-inflammatory / antioxidant drugs and antifibrotic drugs, thereby achieving temporal regulation of inflammatory response / oxidative stress and MMT process, blocking the key pathological cascade of adhesion formation, and fundamentally solving the problem of adhesion prevention and treatment.

[0024] (3) A multifunctional nanocomposite biomaterial platform was established, which achieved good physical barrier performance and synergistic drug regulation based on pathogenesis.

[0025] (4) This nanocomposite hydrogel combines the hydrophilicity of hydrogels with the drug encapsulation advantages of nanomaterials, providing an innovative solution for hydrogel delivery of hydrophobic drugs. Attached Figure Description

[0026] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

[0027] Figure 1 The NMR spectrum of HA-ADH in Example 1 is shown.

[0028] Figure 2 The NMR spectrum of PF127 with dual-terminal aldehydes in Example 2 is shown.

[0029] Figure 3 The particle size distribution diagrams are for (A) blank PF127 micelles and (B) PF127@PFD in Example 3.

[0030] Figure 4 The NMR spectrum of PLA in Example 4 is shown.

[0031] Figure 5 Example 5 shows (A) the scavenging ability of different concentrations of α-LA, DHLA, and PLA for ·OH; and (B) the scavenging ability of different concentrations of PLA and DHLA for H2O2.

[0032] Figure 6 The values ​​of G′, G″, and Tanδ are given for the hydrogels obtained by mixing equal volumes (1:1) of PF127 micelles (25wt%, 20wt%, 15wt%, and 10wt%) and HA-ADH (3.125wt%, 2.5wt%, 1.5wt%, and 1.0wt%) in Example 6; and for the hydrogels obtained by mixing different volume ratios (PF127:HA-ADH = 1:3, 1:2, 1:1, 2:1, and 3:1) under the condition of fixed mass concentrations of PF127 (20wt%) and HA-ADH (2.5wt%).

[0033] Figure 7 The following are examples from Example 7: (A) Strain oscillation scans of different groups of hydrogels; (B) Shear rate scans of different groups of hydrogels; and (C) Strain step experiments of different groups of hydrogels (strain changes alternately from 1% to 500% at 100s intervals).

[0034] Figure 8 The images shown are: (A) Strain oscillation scan of P / H / PLA@PFD before and after H2O2 treatment; (B) Scanning electron micrographs of P / H / PLA@PFD before and after H2O2 treatment; and (C) Cumulative release curves of P / H / PLA@PFD in solutions containing or without H2O2.

[0035] Figure 9 The values ​​represent the quantitative values ​​of the adhesion strength between different groups of hydrogels and rat abdominal wall tissue in Example 9.

[0036] Figure 10 This study investigated the ability of different groups of the original drug and hydrogel to clear intracellular ROS in inflammatory macrophages in Example 10.

[0037] Figure 11 The relative expression levels of macrophage inflammatory-related mRNAs regulated by different groups of the original drug and hydrogel in Example 11 are: (A) TNF-α and (B) IL-6.

[0038] Figure 12 The relative expression levels of different groups of original drugs and hydrogels in PRMC mesenchymal transition-related mRNAs were shown in Example 12: (A) E-Cadherin; (B) Col-1.

[0039] Figure 13 This is an adhesion score chart of the different treatment groups in Example 13 on the 7th day after surgery. Detailed Implementation

[0040] The preferred embodiments of the invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the invention.

[0041] The method involved in this invention is known in the art, and the reagents used in the experiment are commercially available.

[0042] Example 1: Synthesis of adipic acid dihydrazide-functionalized polysaccharides

[0043] Weigh 1.00 g of polysaccharide (any one of hyaluronic acid, chondroitin sulfate, fucoidan, or sodium alginate) and 2.00 g of ADH, and dissolve them in 60 mL of pure water. Stir thoroughly until completely dissolved. Adjust the pH of the reaction solution to 4.75 with 0.1 M hydrochloric acid, then add EDCI to initiate the reaction. Stir at room temperature for 8–24 h, maintaining the pH of the reaction solution at approximately 4.75 during the reaction. After the reaction is complete, adjust the pH to neutral with 0.1 M NaOH solution to terminate the reaction. Dialyze the reaction solution with pure water for 3 days, and freeze-dry to obtain the product. The product is then processed through… 1 The structure was confirmed by H NMR.

[0044] The reaction route diagram for hyaluronic acid functionalized with adipic acid dihydrazide (HA-ADH) is as follows:

[0045]

[0046] Example 2: Synthesis of aldehyde-functionalized Pluronic F127 (PF127)

[0047] First, 12.60 g (1.00 mmol, 1 eq) of dried Pluronic F127 and 2.28 mL (20.00 mmol, 20 eq) of triethylamine were weighed and dissolved in 100 mL of anhydrous dichloromethane. Then, 7.63 g (40.00 mmol, 40 eq) of p-toluenesulfonyl chloride was added, and the mixture was stirred at room temperature for 2 days under N2 protection. Subsequently, the mixture was washed twice with dilute hydrochloric acid and twice with saturated NaHCO3, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and recrystallized from anhydrous diethyl ether to obtain a sulfonated intermediate. Then, 12.00 g (0.95 mmol, 1 eq) of this intermediate was dissolved in 50 mL of anhydrous N,N-dimethylformamide. After dissolution, 2.32 g (19.00 mmol, 20 eq) of p-hydroxybenzaldehyde and 2.63 g (19.00 mmol, 20 eq) of potassium carbonate were added, and the mixture was stirred at 80 °C for 3 days under N2 protection. The product was then diluted with pure water, extracted three times with dichloromethane, and the dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and recrystallized from anhydrous diethyl ether to give a light white solid, PF127. This product was then subjected to… 1 The structure was confirmed by H NMR.

[0048] The synthetic reaction route of HF127 is as follows:

[0049]

[0050] Example 3: Synthesis and Characterization of HF127 Nanomicelles Encapsulated with Antifibrotic Drugs

[0051] Drug-loaded micelles and blank HF127 micelles were prepared using a thin-film dispersion method. A certain amount of pirfenidone (PFD) and PF127 were weighed and added to a 10 mL round-bottom flask, along with an appropriate amount of chloroform, and dissolved by sonication. The organic solvent was evaporated under reduced pressure at 40 °C for approximately 30 min to obtain a PF127@PFD film. A measured volume of preheated double-distilled water was then added, and the mixture was hydrated at 100 rpm for a certain time. The dispersion was then filtered through a 0.45 μm aqueous microporous membrane to remove insoluble free PFD crystals, yielding a clear and transparent drug-loaded micelle solution. The preparation of blank PF127 micelles followed the same procedure. The particle size distribution of PF127@PFD micelles and PF127 micelles was determined using a laser particle size analyzer.

[0052] Drug-loaded nanomicelle solutions containing nintedanib, cryptotanshinone, tanshinone, curcumin, and baicalin were prepared using the same method.

[0053] Experimental results show that ( Figure 3AB): The particle sizes of blank nanomicelles PF127 and drug-loaded nanomicelles PF127@PFD are 15.4 nm and 20.9 nm, respectively, and both show a single-peak distribution, indicating that the micelles were successfully prepared and have good dispersibility.

[0054] Example 4: Synthesis of PLA, a reactive oxygen species-responsive dihydrolipoic acid prodrug containing 2-formylphenylboronic acid

[0055] First, 2.00 g (4.71 mmol, 1 eq) of PB(p)-Br and 0.467 g (2.24 mmol, 0.48 eq) of dihydrolipoic acid were weighed and dissolved in 20 mL of acetonitrile. Then, 0.45 g (11.25 mmol, 2.4 eq) of sodium hydroxide was added. The mixture was stirred at room temperature for 24 h under N2 protection. After the reaction was completed, the solution was concentrated under reduced pressure and purified by column chromatography to obtain a yellow oily liquid, 2PB(p)-LA.

[0056] Next, 2PB(p)-LA (1.05 mmol, 1 eq) and methylboric acid (10.49 mmol, 10 eq) were dissolved in a certain amount of dichloromethane, followed by the addition of trifluoroacetic acid (approximately 5% of the total volume of the reaction solution), and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, redissolved in ethanol, dialyzed against ethanol / water (1:1, v / v) for 1 day, and then dialyzed against pure water for another 1 day. The product was then freeze-dried to obtain a pale yellow solid PLA. 1 The structure was confirmed by H NMR.

[0057] The synthetic reaction route of PLA is as follows:

[0058]

[0059] Example 5: In vitro reactive oxygen species scavenging capacity of PLA

[0060] ·OH removal

[0061] The scavenging ability of dihydrolipoic acid prodrug (PLA), dihydrolipoic acid (DHLA), and α-lipoic acid (α-LA) for ·OH was investigated using the Fenton degradation method for methylene blue. PLA, DHLA, and α-LA at final concentrations of 12.5, 25, 50, and 100 μM were reacted with 100 μM Fenton's solution (·OH generated from a mixture of 100 μM H₂O₂ and 100 μM FeCl₂), followed by the addition of MB (7.5 μg / mL) for color development. The specific sample addition procedure is as follows:

[0062] Sample group: 1 mL of sample solutions of different concentrations (50, 100, 200, 400 μM) + 1 mL of FeCl2 (400 μM) + 1 mL of H2O2 (400 μM) + 1 mL of MB (30 μg / mL)

[0063] Positive control group: 1 mL deionized water + 1 mL FeCl2 (400 μM) + 1 mL H2O2 (400 μM) + 1 mL MB (30 μg / mL)

[0064] Negative control group: 3 mL deionized water + 1 mL MB (30 μg / mL)

[0065] Different drugs were mixed with Fenton's solution, and then MB solution was added for colorimetric analysis. The entire reaction was carried out in an aqueous system under light-protected conditions. After sample addition, the mixture was incubated for 30 minutes, and the absorbance of each sample at 664 nm was measured using a UV-Vis spectrophotometer. The ·OH scavenging rate was calculated as follows:

[0066]

[0067] A X A0 represents the absorbance value corresponding to the positive control group, and A1 represents the absorbance value corresponding to the negative control group.

[0068] H2O2 removal

[0069] The scavenging ability of PLA and DHLA for H2O2 was indirectly investigated using the Fenton degradation methylene blue method. PLA and DHLA at final concentrations of 25 and 50 μM were pre-incubated with 100 μM H2O2 for 0, 15, 30, 45, 60, 120, 240, 360, and 480 min, respectively, before adding 100 μM FeCl2 and MB (7.5 μg / mL) for color development. The negative and positive control groups were prepared using the same method as the ·OH scavenging experiment. After adding samples, all samples were incubated for 30 min, and the absorbance at 664 nm was measured using a UV-Vis spectrophotometer. The H2O2 scavenging rate was calculated as follows:

[0070]

[0071] A X The absorbance values ​​for different sample groups at various time points are: A0 is the absorbance value for the positive control group, and A1 is the absorbance value for the negative control group.

[0072] Experimental results show that ( Figure 5(AB): PLA, DHLA, and α-LA at different concentrations all effectively inhibited the degradation of methylene blue, and the inhibitory effect increased in a dose-dependent manner. This indicates that PLA, DHLA, and α-LA can all scavenge the ·OH generated in the system. α-LA showed the strongest ·OH scavenging ability due to its strongly electrophilic disulfide five-membered ring structure, which allows it to react with free radicals. DHLA has a reduced thiol structure, thus also exhibiting a strong ·OH scavenging ability. The weaker scavenging ability of PLA compared to α-LA and PLA is mainly attributed to the shorter incubation time; PLA requires a certain amount of time to respond to ROS bond breaking, thus exhibiting a relatively weaker scavenging effect.

[0073] After PLA and DHLA were pre-incubated with H2O2 for different times, Fe was added. 2+ The residual H2O2 content in the system was detected by colorimetric reaction with MB solution, indirectly examining the scavenging ability of PLA and DHLA for H2O2. Figure 5 As shown in Figure B, 25 μM and 50 μM DHLA could only remove 25% and 50% of H2O2 (100 μM), respectively, and the removal rate tended to stabilize without significant change over time. With prolonged exposure time, the ability of PLA to remove H2O2 continuously increased, with 50 μM PLA achieving a removal rate of 96.8% for H2O2 at 8 hours, significantly higher than DHLA. This is because PLA, upon responding to H2O2, removes it through a two-step reaction cascade involving bond breaking and drug release, demonstrating a stronger ROS removal capacity and providing a basis for subsequent mitigation of oxidative stress.

[0074] Example 6: Preparation and Characterization of Nanocomposite Hydrogel Formulations

[0075] HA-ADH solutions with concentrations of 3.125, 2.5, 1.875, and 1.25 wt% were prepared, along with drug-loaded micelle solutions with Pluronic F127 concentrations of 10, 15, 20, and 25 wt%. Solid PLA was weighed and added to the drug-loaded micelle solutions, controlling the PLA concentration to be 0.1-0.5 wt%. Maintaining a fixed mass concentration ratio of HA-ADH solution to micelle solution of 1:8, the HA-ADH solution and drug-loaded micelle solutions were mixed at volume ratios of 8:1-1:4 to obtain nanocomposite hydrogel formulations with different crosslinking ratios. The mechanical strength of representative nanocomposite hydrogels with different crosslinking ratios was then investigated.

[0076] The same method was used to prepare a nanocomposite hydrogel formulation with chondroitin sulfate, brown algae polysaccharide, and sodium alginate as polysaccharide chains.

[0077] Experimental results show that ( Figure 6(AB): The higher the mass concentration of the polymer, the higher the mechanical strength. The G′ value of the gel formed by a 250:31.25 ratio is approximately 2545 Pa. The G′ value of the gel formed by a 200:25 ratio is between 1000-1100 Pa, close to the mechanical strength of abdominal organs. By fixing the mass concentrations of PF127 and HA-ADH and changing the volume ratio, when the volume ratio of PF127 to HA-ADH is 1:1, the G′ value of the P / H gel remains around 1100 Pa, exhibiting mechanical strength relatively close to that of abdominal organs.

[0078] Example 7: Investigation of the rheological properties of the nanocomposite hydrogel formulation P / H / PLA@PFD

[0079] A hydrogel precursor solution with an HA-ADH concentration of 2.5 wt% and a drug-loaded micelle concentration of 20 wt% (using PFD as an example) was prepared, with PLA mixed in the drug-loaded micelles at a mass concentration of 0.3 wt%. The hydrogel (P / H / PLA@PFD) prepared by mixing the HA-ADH solution and the drug-loaded micelle solution in equal volumes was then further investigated.

[0080] The basic parameters of the rheometer were set as follows: the measuring fixture was a CP20 cone plate with a gap of 1 mm, the test temperature was 25℃, and the air pressure was 5 bar. The mechanical strength of hydrogels with different proportions was investigated by strain scanning (frequency 1 Hz, strain 0.1%-100%). Furthermore, the shear thinning and self-healing abilities of the hydrogels were investigated by shear rate scanning and strain step scanning.

[0081] Experimental results show that ( Figure 7 AC): Both drug-loaded and undrug-loaded hydrogels maintained a relatively stable G′ > G″ within a strain range of 0.1-100%, demonstrating that the gel system formed by PF127 and HA-ADH possesses excellent solid-like properties. Compared to P / H gel (1000-1100 Pa), the G′ value of P / H@PFD gel increased to 1300-1400 Pa. This is because the introduction of PFD enhanced the hydrophobicity of the hydrophobic regions of PF127 micelles, making the internal network of the gel more compact and exhibiting a slight increase in mechanical strength. Meanwhile, P / H / PLA gel and P / H / PBA@PFD gel have larger G′ values. This is because the 2-FPBA and HA-ADH hydrazide groups of PLA are rapidly cross-linked through click chemistry, enhancing the physical entanglement with the P / H gel network and increasing the cross-linking density. The G′ value of P / H / PBA@PFD is maintained at around 2000 Pa, which is slightly higher than the G′ value of abdominal organs (1100 Pa), but its Tanδ (0.368) is close to that of abdominal organs (0.18-0.3), indicating similar viscoelastic behavior. Figure 7A). Subsequently, the relationship between the viscosity of P / H / PLA@PFD and shear rate was further investigated. Within the shear rate range of 0.1-100 s⁻¹, all groups of gels exhibited the characteristic of "shear thinning," providing a basis for the application of hydrogels to wounds via injection. Figure 7 B).

[0082] The self-healing ability of the gel was further characterized by strain step tests. The results showed that when the strain value was 1%, the hydrogel was predominantly elastic (G″ < G′); immediately adjusting the strain value to 500%, the hydrogel modulus immediately reversed, at which point viscosity became dominant (G″ > G′). When the strain was changed back to 1%, the viscoelastic modulus of the hydrogel almost recovered to the value at the first 1% strain. After three cycles, the modulus value remained largely unchanged, indicating that the hydrogel can rapidly recover to its initial state from strain deformation, demonstrating rapid and excellent self-healing properties. Figure 7 C).

[0083] Example 8: Investigation of reactive oxygen species response bond breaking and drug release capacity of nanocomposite hydrogel formulation P / H / PLA@PFD

[0084] The microstructure and mechanical properties of P / H / PLA@PFD gel after treatment with 0.1 mM H2O2 for 48 h were investigated using scanning electron microscopy and rheology. The reactive oxygen species-responsive bond-breaking properties of P / H / PLA@PFD gel were also examined.

[0085] The in vitro release behavior of the drug PFD in P / H / PLA@PFD was investigated by dialysis. The hydrogel was sealed in a dialysis bag (MwCO 1000Da) and immersed in 40 mL of PBS or PBS solution containing 0.1 mM H₂O₂. The release solution was collected at different time points (0.5, 1, 2, 3, 4, 5, 6, and 7 days), and the same volume of fresh release medium was added. The concentration of the release solution was examined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated.

[0086] Experimental results show that ( Figure 8 AC): Under the action of H2O2, the G′ value of P / H / PBA@PFD gel decreased, remaining almost consistent with the mechanical strength (1300 Pa) of P / H@PFD. This demonstrates that the benzodiazepine crosslinking network formed by PLA and HA-ADH broke down in response to H2O2, while the remaining P / H gel network maintained its gel properties. After H2O2 treatment, the linear viscoelastic region of the gel shortened, which may be due to the swelling of the gel network in solution, leading to gel brittleness. Figure 8 A).

[0087] SEM results showed that P / H / PLA@PFD exhibited a dense three-dimensional porous structure with small pore sizes. After H2O2 treatment, the pores of P / H / PLA@PFD were observed to be significantly larger and of uneven size. This indicates that H2O2 disrupted part of the gel structure, reducing its density. It is speculated that the benzodiazepine crosslinking network formed by PLA and HA-ADH broke down in response to H2O2, leaving the P / H gel network to maintain its gel properties. Figure 8 B).

[0088] In vitro release results showed that P / H / PLA@PFD slowly and continuously released PFD in PBS medium. However, in the presence of 0.1 mL H₂O₂, the cumulative release rate of P / H / PLA@PFD changed significantly in the first 2 days, increasing from 10% in blank PBS medium to 37%. This is because the benzodiazepine crosslinking network formed by PLA and HA-ADH broke down under the action of H₂O₂, reducing the gel crosslinking density and thus accelerating PFD release. These results indicate that P / H / PLA@PFD gel exhibits ROS-responsive drug release behavior. Figure 8 C).

[0089] Example 9: Investigation of tissue adhesion of nanocomposite hydrogel formulation

[0090] The adhesion of hydrogel to rat abdominal wall tissue was investigated using an overlap shear test. First, freshly prepared rat abdominal wall tissue was cut into uniform 2.5cm × 2.0cm slices. 400μL of the prepared hydrogel was placed on the rat abdominal wall, spread evenly, and then covered with another layer of abdominal wall tissue of uniform thickness and size. The overlap area was maintained at 2.5cm × 1.0cm. The resulting samples were allowed to stand at room temperature for 5 minutes to allow for sufficient adhesion between the gel and the tissue. Finally, an electronic universal testing machine was used to clamp the assembled samples in upper and lower clamps and test the samples at a speed of 50mm / min. The adhesive shear force (F / N) and displacement of each sample were recorded. The formula for calculating the hydrogel adhesive force (kPa) is: maximum adhesive shear force / overlap area.

[0091] Experimental results show that ( Figure 9 The adhesion strength of P / H gel to the rat abdominal wall, measured by the overlap shear test, was approximately 1.68 kPa, which was not significantly different from that of commercially available HA gel. Compared to P / H and P / H@PFD, the adhesion strength of P / H / PLA and P / H / PLA@PFD was significantly improved, reaching 2.2 and 2.5 kPa, respectively, showing improved adhesion compared to commercially available HA gel. This is because the introduction of small molecule PLA provides more free carboxyl groups, which enhances adhesion to tissues through hydrogen bonding and other interactions.

[0092] Example 10: Investigation of the intracellular ROS scavenging ability of nanocomposite hydrogel formulation

[0093] LPS-induced high intracellular oxidative stress in RAW 264.7 cells was used as a positive model to investigate the hydrogel's ability to scavenge intracellular ROS. First, RAW 264.7 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 RAW 264.7 cells were seeded at a density of 1 cells / well in 12-well plates. After cell adhesion, 1 μg / mL LPS was added for 12 h to induce inflammatory cell formation. The culture medium containing the inducing factor was then discarded, and the cells were washed twice with PBS. The prototype drug control group was incubated with culture medium containing 50 μM DHLA, 2 mM PFD, and 50 μM DHLA + 2 mM PFD for 24 h. The prepared gel was placed on the upper layer of a Transwell permeable cell culture chamber, and 2 mL of complete culture medium was added for incubation with the cells for 24 h. The final drug concentration in the gel group was consistent with that in the prototype drug group. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. 500 μL of serum-free culture medium containing the DCFH-DA probe (10 μM) was added to each well. After incubation in the dark for 30 min, the cells were washed with PBS, digested, collected, centrifuged, resuspended in PBS, and intracellular ROS levels were detected by flow cytometry.

[0094] Experimental results show that ( Figure 11 Both the CT and IL-4 groups showed weak DCF fluorescence. However, after 12 h of induction with 1 μg / mL LPS, the DCF fluorescence intensity significantly increased, indicating that LPS effectively induced oxidative stress in the cells. After LPS-induced cells were treated with 50 μM DHLA for 12 h, the intracellular DCF fluorescence level significantly decreased. PFD could also clear intracellular ROS to some extent, but its effect was not as good as DHLA. The DHLA+PFD group further reduced the DCF fluorescence level, indicating that the combination of PFD and DHLA had a better effect on alleviating oxidative stress. Subsequently, the ability of different groups of gels to clear intracellular ROS was examined. The P / H / PLA@PFD group showed the weakest DCF fluorescence intensity, which was the result of the combined action of PLA cascade ROS clearance, the released small amount of PFD, and the P / H blank gel. The above experiments demonstrate that P / H / PLA@PFD has a significant ROS clearance ability and can effectively alleviate oxidative stress caused by postoperative injury.

[0095] Example 11: Investigation on the intracellular ROS scavenging and anti-inflammatory functions of nanocomposite hydrogel formulations

[0096] RAW 264.7 cells were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) to establish an inflammation model, and the anti-inflammatory ability of the hydrogel was investigated. RAW 264.7 cells were sputtered at 2 × 10⁻⁶ cells / cells. 5 RAW 264.7 cells were seeded at a density of 1 cell / well in six-well plates. After full cell adhesion, 1 μg / mL LPS and 20 ng / mL IFN-γ were added to stimulate the cells to transform into M1 type. After 12 h of stimulation, the culture medium containing the inducing factors was discarded, and the cells were washed twice with PBS. The prototype drug control group was incubated with culture medium containing 50 μM DHLA, 2 mM PFD, and 50 μM DHLA + 2 mM PFD for 24 h. The prepared gel was placed on the upper layer of a Transwell permeable cell culture chamber, and 2 mL of complete culture medium was added and incubated with the cells for 24 h. The final drug concentration in the gel group was consistent with that in the prototype drug group. RAW 264.7 cells were stimulated with 40 ng / mL IL-4 to transform into M2 type cells as a control; untreated cells were classified as M0 type. The anti-inflammatory ability of the hydrogel was assessed by detecting the relative expression levels of IL-6 and TNF-α mRNA in macrophages using RT-PCR.

[0097] Experimental results show that ( Figure 11 (AB): Further investigation into the anti-inflammatory effects of the hydrogels revealed that the DHLA+PFD treatment group synergistically reduced the mRNA expression of pro-inflammatory factors TNF-α and IL-6. Among the hydrogel groups, the P / H / PLA@PFD hydrogel exhibited the best anti-inflammatory effect.

[0098] Example 12: Investigation on the inhibition of mesothelial cell transformation into mesenchyme by nanocomposite hydrogel formulation

[0099] Extraction and separation of RPMCs: Rats weighing 120-160g were euthanized by cervical dislocation and thoroughly sterilized by immersion in 75% alcohol. The tissues were then transferred to a laminar flow hood, and the greater omentum and gastrosplenic ligament were harvested. The tissues were washed with PBS, minced, and transferred to 20 mL of digestion solution (trypsin-EDTA digestion solution (0.25%)). The tissues were incubated at 37°C with shaking for 15 min. After digestion, an equal volume of complete culture medium was added to terminate the digestion, and large tissue pieces were removed by passing the solution through a 100-mesh sieve. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 4 mL of complete rat peritoneal mesothelial cell culture medium, transferred to a culture dish, and incubated statically at 37°C with 5% CO2.

[0100] RPMC is set at 1×10 5Cells were seeded at a density of [number] cells / well in six-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. The control group received no treatment, while the control group was stimulated with 10 ng / ml TGF-β1 to induce MMT in RPMCs. The prototype drug control group was co-incubated with culture medium containing 50 μM DHLA, 2 mM PFD, and 50 μM DHLA + 2 mM PFD, along with 10 ng / ml TGF-β1, for 72 hours. The gel group was co-incubated with 10 ng / ml TGF-β1 in the upper layer of a Transwell permeable cell culture chamber for 72 hours, with the final concentration consistent with the prototype drug group. The relative expression levels of E-cadherin and Col-1 mRNA in RPMCs were detected by RT-PCR to assess the hydrogel's ability to inhibit MMT.

[0101] Experimental results show that ( Figure 12 (AB): Under the induction of 10 ng / mL TGF-β1, the mRNA level of E-cadherin (epithelial phenotype marker) in RPMCs was significantly downregulated, while the mRNA level of Col-1 (fibroblast phenotype marker) was significantly upregulated, indicating that TGF-β1 successfully induced MMT in RPMCs. Under the treatment of 2 mM PFD, the mRNA expression level of E-cadherin in RPMCs was significantly upregulated, while the mRNA level of Col-1 was significantly downregulated, consistent with the Western blot results, indicating that PFD can effectively reverse the MMT process in RPMCs. DHLA had no significant effect on the mRNA levels of E-cadherin and Col-1. Both P / H@PFD and P / H / PLA@PFD gel treatments increased the mRNA expression level of E-cadherin and decreased the mRNA level of Col-1, but the effect was not as good as that of free PFD, which may be due to the gel network slowing down the release of PFD.

[0102] Example 13: In vivo anti-adhesion efficacy study of nanocomposite hydrogel formulation

[0103] A rat cecal-abdominal wall abrasion model was constructed to investigate the in vivo anti-adhesion effect of a nanocomposite hydrogel formulation. First, SD rats that had been normally fed for one week were randomly divided into 7 groups (n=6 per group): ① sham surgery group, ② model group, ③ commercially available sodium hyaluronate (HA) group, ④ blank gel P / H group, ⑤ P / H / PLA gel loaded with prodrug, ⑥ P / H@PFD group loaded with antifibrotic drug, and ⑦ final gel P / H / PLA@PFD group. The rats were anesthetized and shaved. The surgical area was disinfected with ethanol. An incision (approximately 4 cm long) was made along the midline of the abdomen to expose the cecum. The cecum was rubbed back and forth with sterile medical gauze until blood spots appeared on the cecal surface. Simultaneously, an injury area of ​​approximately 1 cm × 2 cm was cut into the abdominal wall (including the peritoneum and muscle layer) on the opposite side of the cecum to form a damaged abdominal wall. The Model group received no medication and the cecum was directly repositioned. The HA group received an injection of commercially available sodium hyaluronate at the injury site followed by cecum repositioning. The self-made gel group received hydrogel injected into both the damaged cecum and abdominal wall surface, and the cecum was carefully repositioned. The Sham group underwent no abrasion, but the procedure was the same as the Model group. All groups then had the inner abdominal wall sutured with 4-0 sutures and the outer skin sutured with 3-0 sutures. Finally, the sutured areas were wiped with cotton balls containing 0.1 wt% ampicillin to prevent infection, and the rats were returned to their cages according to their groups, with free access to water and food. On postoperative day 7, six rats were randomly selected from each group, euthanized, and their abdominal cavities were opened to observe and photograph the adhesions within the abdominal cavity. A standard scoring system was used to score the adhesions in each group.

[0104] Experimental results show that ( Figure 13 In the Sham group, no adhesions were observed, with a score of 0; while in the Model group, large and severe adhesions between the cecum and the abdominal wall were observed, with an overall score of 4.83. Comparing the adhesion prevention effects of different gel groups, commercially available HA gel showed some anti-adhesion ability (score close to 4). Compared to the HA group, the average score of the P / H / PLA gel group was 2. Ultimately, the P / H / PLA@PFD gel group showed the lowest adhesion score (0.4). This is because P / H / PLA@PFD not only possesses excellent physicochemical properties (suitable mechanical strength, self-healing ability, and tissue adhesion), but also regulates the high oxidative stress and inflammatory environment of the peritoneal microenvironment. Upon responding to ROS, it accelerates the release of PFD to regulate fibrosis, combining physical barriers and drug regulation to act on all key stages of PAA development.

Claims

1. A sequential-release drug anti-adhesion nanocomposite hydrogel formulation, characterized in that, The hydrogel formulation is constructed in the following manner: a) A gel network basic structure is formed by cross-linking adipate dihydrazide-functionalized polysaccharide with drug-loaded nanomicelles via dynamic acylhydrazone bonds; b) Introduce PLA (2-FPBA-SRS-FPBA-2, where SRS represents dihydrolipoic acid and 2-FPBA is 2-formylphenylboronic acid), a reactive oxygen species responsive dihydrolipoic acid prodrug, into the basic structure of the gel network. Benzodiazepine is formed through a click chemical reaction between 2-FPBA on the molecule and the acylhydrazide group derived on the polysaccharide chain, thereby strengthening the crosslinking density of the gel network and endowing the gel with reactive oxygen species responsive sequential drug release function. The drug-loaded nanomicelles are composed of aldehyde-functionalized Pluronic F127 (PF127) encapsulating anti-fibrotic drugs; the sequential drug release occurs when the hydrogel is in a high-level reactive oxygen species environment, the reactive oxygen species cleave the benzodiazepine bond to release the antioxidant drug dihydrolipoic acid, and the pore size of the gel network increases accordingly, accelerating the release of the anti-fibrotic drug.

2. The sequential-release drug anti-adhesion nanocomposite hydrogel formulation according to claim 1, characterized in that, The polysaccharide is any one of hyaluronic acid, chondroitin sulfate, fucoidan, and sodium alginate; the antifibrotic drug is any one of pirfenidone, nintedanib, cryptotanshinone, tanshinone, curcumin, and baicalein.

3. The sequential-release drug anti-adhesion nanocomposite hydrogel formulation according to claim 1, characterized in that, The nanomicelles have a particle size distribution in the range of 0-50 nm, a drug encapsulation efficiency of ≥60%, and a drug loading of ≥0.01 wt%. The aldehyde functionalization degree of PF127 is 2 aldehyde groups / PF127 molecule. The degree of substitution of adipic acid dihydrazide in the adipic acid dihydrazide functionalized polysaccharide is 30-80%, and the molecular weight ranges from 10-500 kDa.

4. A sequential-release drug anti-adhesion nanocomposite hydrogel formulation according to any one of claims 1-3, characterized in that, The preparation steps include the following; Step 1: Synthesize adipic acid dihydrazide-functionalized polysaccharides through an amide condensation reaction between the carboxyl groups on the polysaccharide chain and adipic acid dihydrazide; Step 2: Synthesize PF127 modified with aldehydes at both ends and prepare PF127 drug-loaded nanomicelles; Step 3: Synthesize PLA, a reactive oxygen species-responsive dihydrolipoic acid prodrug molecule with 2-formylphenylboronic acid grafted with thiol groups at both ends; Step 4: Mix PLA with PF127 drug-loaded nanomicelle solution to prepare mixed precursor solution, and mix the mixed precursor solution with adipic acid dihydrazide functionalized polysaccharide solution to obtain sequential release drug anti-adhesion nanocomposite hydrogel formulation.

5. The preparation method of a sequential-release drug anti-adhesion nanocomposite hydrogel formulation according to claim 4, characterized in that, The concentration of the adipic acid dihydrazide-functionalized polysaccharide solution before mixing is 1-5 wt%, and the concentration of the PF127 drug-loaded nanomicelle solution is 5-25 wt%; the concentration of the PLA solution is 0.1-0.5 wt%; the mixing mass concentration ratio of the adipic acid dihydrazide-functionalized polysaccharide solution to the PF127 nanomicelle solution is 8:1-1:4; the gel precursor solution is mixed by pre-mixing or partial mixing.

6. The use of a sequential-release drug-resistant anti-adhesion nanocomposite hydrogel formulation according to any one of claims 1-5 in the preparation of a barrier material for preventing postoperative tissue adhesion.

7. The use according to claim 6, characterized in that, The postoperative tissue adhesion type is any one or more of abdominal adhesion, pelvic adhesion, and uterine adhesion.

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