Cyclodextrin molecular bridges enhance ros-responsive thermosensitive hydrogels and applications thereof

Abstract

The application discloses a cyclodextrin molecular bridge enhanced ROS response temperature-sensitive gel and application thereof. The gel is composed of a temperature-sensitive base material Pluronic F127, a supramolecular modifier sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) and a ROS response prodrug. The SBE-beta-CD serves as a "molecular bridge", and through hydrophobic inclusion of the F127 hydrophobic segment and reconstruction of a hydrogen bond network, an enhanced gel network is formed, the gelation temperature is accurately reduced to 27.4 DEG C, the critical strain is increased to 0.1428, room temperature operation, rapid gelation at nasal physiological temperature and high shear resistance are realized. The ROS response prodrug is DHA-PEG 2k -TK-Insulin, and oxidation stress triggered rupture is realized through a thio-ketone (TK) bond. The application constructs a "physical interception-chemical trigger" dual intelligent controlled release architecture, and solves the problems of drug burst release and mucosal retention. The composite hydrogel can be used as a non-invasive delivery carrier for protein and polypeptide drugs (such as insulin) in the nasal cavity, and has the significant advantages of precision, long-acting and intelligent drug release.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary technical field of biomedical polymer functional materials and intelligent drug delivery systems, specifically relating to a ROS-responsive in-situ thermosensitive composite hydrogel system based on supramolecular "molecular bridge" network reconstruction and its application. Background Technology

[0002] Currently, clinical administration of protein drugs (such as insulin) is still mainly by injection, which has problems such as poor patient compliance and significant local adverse reactions. Nasal administration, as a non-invasive delivery route, provides an important direction for non-injection administration of protein drugs, but its clinical application urgently requires the development of intelligent delivery carriers that can simultaneously solve problems such as easy enzymatic degradation of drugs, poor mucosal retention, and uncontrollable release behavior.

[0003] Thermosensitive hydrogels have become a research hotspot for nasal drug delivery carriers due to their combination of room temperature injectability / sprayability and in-situ gelation at physiological temperatures. Among them, Pluronic F127 is the most representative thermosensitive material, but it has three inherent defects: (1) The rheological behavior is mismatched with physiological temperature. High-concentration systems tend to gel prematurely, leading to injection obstruction, while low-concentration systems gel too slowly at the physiological temperature (~35℃) of the nasal cavity, with the gel point measured at 29.4005℃; (2) The microscopic topological structure is unstable. Due to the lack of strong interaction support, the critical strain is only 0.1108, making it prone to disintegration under physiological shear forces such as mucosal ciliary movement; (3) Lack of strength. In vitro release experiments using methylene blue as a model drug showed that, under deionized water medium and constant temperature oscillation at 37℃, the drug release rate of pure F127 gel was close to 100% within 6 hours, and the gel system itself disintegrated in the medium. This phenomenon indicates that the structural stability of pure F127 gel under these conditions is poor, and it cannot form a long-term drug reservoir.

[0004] Existing research mainly focuses on HP-β-CD, but its competitive hydration effect significantly increases the gelation point of F127, disrupting the micelle self-assembly network and creating a technical stalemate—existing solutions cannot simultaneously achieve rapid and stable gelation at nasal cavity temperatures. Previous research by the inventors revealed that sulfobutyl ether-β-cyclodextrin (SBE-β-CD) exhibits a unique "supramolecular molecular bridge" effect at specific low proportions: on the one hand, it partially encapsulates the polypropoxy segments of F127 through hydrophobic cavities; on the other hand, it reconstructs the hydrogen bond network of the system through sulfonic acid groups (infrared spectroscopy shows the shift of characteristic peaks of COC and OH). This not only lowers the gelation point from 29.4005℃ ​​to 27.4℃ but also significantly improves the abundance of micelle aggregation and the complex shear modulus, achieving reverse simultaneous optimization of gelation temperature and mechanical strength, breaking through existing performance bottlenecks.

[0005] Furthermore, existing technologies mostly rely on single-dimensional "spatial positioning" drug delivery, lacking "on-demand response" to oxidative stress-related pathological environments. While there are independent studies on ROS-responsive prodrugs, no reports have yet documented a deep coupling of the molecular breakage mechanism of ROS-responsive protein prodrugs with the physical barrier mechanism of modified thermosensitive gels to construct a dual-synergistic "physical interception-chemical triggering" intelligent drug delivery architecture. This architecture could simultaneously solve the industry-wide common challenges of "difficulty in mucosal retention" and "difficulty in precise release" in nasal drug delivery.

[0006] Therefore, developing an SBE-β-CD / F127 composite hydrogel system that combines suitable gelation temperature, high mechanical strength, strong corrosion resistance, and ROS-responsive precise drug release characteristics for non-invasive sustained-release delivery of protein and peptide drugs through the nasal cavity has significant clinical implications and industrial application value. Summary of the Invention

[0007] Existing technical problems: (1) Severe mismatch between thermosensitive behavior and physiological window for nasal administration: Pure F127 at concentrations of 24% (w / w) and above completely gels at room temperature, significantly limiting injectability / sprayability; while low concentration systems gel slowly at physiological temperatures of ~35℃ in the nasal cavity, failing to achieve instantaneous in-situ fixation and long-term retention of the drug on the mucosal surface.

[0008] (2) The F127 supramolecular network topology has intrinsic defects: F127 molecules aggregate only through weak hydrophobic interactions, with a critical strain of only 0.1108, resulting in extremely poor shear resistance. In vitro release experiments using methylene blue as a model drug showed that, under deionized water medium and isothermal oscillation at 37°C, the pure F127 gel exhibited a nearly 100% methylene blue release rate within 6 hours due to its loose network structure, confirming that its three-dimensional network lacks an effective physical barrier and is unable to resist water molecule penetration and gel erosion. It completely lacks a physical protective barrier for long-term sustained release.

[0009] (3) Traditional cyclodextrin modification is trapped in a technical deadlock of “negative effect”: Commonly used modifiers such as hydroxypropyl-β-cyclodextrin (HP-β-CD) disturb the hydration hydrogen bond network of the PEO chain of F127 due to competitive hydration effect. After modification, the gelation point is increased instead, and the reverse regulation dilemma of “increased gelation point and decreased strength after modification” is trapped, which cannot simultaneously meet the dual indicators of low temperature gelation and high strength gel.

[0010] (4) The drug delivery carrier lacks the "intelligent judgment" dimension of pathological microenvironment response: The existing F127-based thermosensitive gel only has the "spatial positioning" function of drug delivery in the nasal cavity. It lacks the ability to release drugs in response to the oxidative stress (ROS) microenvironment and cannot achieve on-demand and precise drug release based on the ROS signal at the lesion site, which is seriously out of touch with the clinical precision treatment needs.

[0011] (5) The core common problem of non-invasive nasal delivery of protein drugs has not been solved: Existing carriers cannot simultaneously solve the industry pain points of "not being able to stay" (poor retention), "not being tolerated" (weak resistance to shearing / dissolution) and "not being able to control accurately" (irregular release and no targeting) on ​​the nasal mucosa surface, which has become a key technical barrier for non-invasive nasal delivery of large molecular protein drugs such as insulin.

[0012] The key issues to be addressed: (1) Construction of SBE-β-CD-mediated "supramolecular molecular bridge" enhanced gel network: 3% SBE-β-CD was precisely screened as a functional modifier, utilizing its selective inclusion effect on the hydrophobic PPO segments of F127 and the hydrogen bond network reconstruction effect of the system (infrared verification showed that the COC increased from 1085.65 cm⁻¹). - ¹Displaced to 1085.57 cm - ¹, OH is 3491.84 cm - ¹Displaced to 3496.46cm - ¹), the gelation temperature of F127 was precisely adjusted from 29.4005℃ ​​to 27.42379℃, which not only avoids the problem of premature gelation at room temperature, but also perfectly matches the physiological temperature of 35℃ in the nasal cavity; at the same time, the critical strain was increased from 0.1108 to 0.1428, and the complex shear modulus was significantly improved, achieving a leapfrog improvement in shear / dissolution resistance, and completely breaking through the technical deadlock of traditional modification "heating and reducing strength".

[0013] (2) Constructing a high-coupling-rate ROS-responsive insulin prodrug and achieving deep gel coupling: chemically synthesized DHA-PEG 2k The TK-insulin prodrug ensures efficient coupling between insulin and a polymeric carrier, utilizing the high specificity of the TK bond to ROS to trigger cleavage under oxidative stress. Traditional lipid-soluble or water-soluble drugs, after nasal administration, struggle to effectively cross the nasal mucosal barrier to reach the brain, resulting in low brain bioavailability. This study innovatively designed the amphiphilic prodrug molecule DHA-PEG-TK-Insulin, which not only possesses excellent mucosal permeability but also demonstrates the potential to directly enter the brain via nasal epithelial cells, bypassing the blood-brain barrier.

[0014] (3) Establish a dual intelligent controlled release architecture of "physical interception-chemical triggering": relying on the dense three-dimensional network of SBE-β-CD / F127 composite gel to form a strong physical barrier, intercepting the rapid diffusion of drugs and completely eliminating the problem of drug burst release of pure F127 gel (the release rate is significantly delayed from ~100% in 6 hours); by utilizing the specific breakage of TK bond in the ROS pathological microenvironment, the chemical triggering of active insulin is achieved to achieve precise drug release, so that the carrier has the intelligent judgment ability of "physiological environment stability and lesion environment drug release".

[0015] (4) Systematically overcome common industry problems in non-invasive nasal delivery of protein drugs: Through the above technical means, construct an all-purpose drug delivery carrier that combines room temperature injectability / sprayability, in-situ fixation performance with rapid gelation at 35°C in the nasal cavity, high mechanical strength with shear / dissolution resistance, and precise controlled release performance in response to the ROS microenvironment, fundamentally solving the core problems of drugs "not being retained, not being tolerated, and not being accurately controlled" in the nasal mucosa.

[0016] (5) Filling the technological gap of "intelligent response + thermosensitive gel" synergistic drug delivery: Realizing the deep synergy between ROS-responsive prodrug chemical controlled release and thermosensitive composite gel physical controlled release, providing a non-invasive, efficient and precise nasal drug delivery technology solution for large molecular drugs such as insulin and other protein peptides, filling the technological gap of synergistic drug delivery systems in this field.

[0017] To address the shortcomings of existing technologies, this invention provides a highly effective cyclodextrin-based molecular bridge-enhanced ROS-responsive thermosensitive gel and its applications. This invention utilizes sulfobutyl ether-β-cyclodextrin (SBE-β-CD) to enhance the microstructure and reconstruct the hydrogen bond network of Pluronic F127 micelles, constructing a multifunctional drug delivery platform with high mechanical strength, corrosion resistance, and oxidative stress-triggered degradation characteristics. This platform can serve as a non-invasive or minimally invasive sustained-release carrier for large molecule drugs such as proteins and peptides.

[0018] Addressing the four core technical shortcomings of existing Pluronic F127 thermosensitive hydrogels: Mismatch between thermosensitive behavior and physiological window: Pure F127 at concentrations of 24% (w / w) and above forms a complete gel at room temperature, significantly limiting injectability / sprayability; while at low concentrations, the gelation rate is slow at the physiological temperature of ~35°C in the nasal cavity, making it impossible to achieve instantaneous in-situ fixation and long-term retention of the drug on the mucosal surface.

[0019] The gel network topology is inherently fragile: the molecules aggregate only through weak hydrophobic interactions, the critical strain is only 0.1108, and it is very easy to shear disintegrate under the beating of nasal cilia and the flushing of body fluids, resulting in a drug burst release of nearly 100% within 6 hours, lacking physical barrier efficacy; Traditional modification strategies are stuck in the bottleneck of "increasing temperature and decreasing strength": commonly used modifiers (such as HP-β-CD) disturb the hydrogen bond network due to competitive hydration effect, causing the gelation temperature to rise instead of fall (to above 29.4005℃), and cannot simultaneously achieve low-temperature gelation and strength improvement. The lack of response dimension in the pathological microenvironment: the carrier only has spatial positioning function and lacks the ability to identify and logically respond to oxidative stress (ROS) signals, thus failing to achieve precise and on-demand drug release.

[0020] Traditional lipid-soluble or water-soluble drugs, when administered nasally, have difficulty effectively crossing the nasal mucosal barrier to reach the brain, resulting in low brain bioavailability. This study innovatively designed the amphiphilic prodrug molecule DHA-PEG-TK-Insulin, which not only possesses excellent mucosal permeability but also demonstrates the potential to cross the epithelial cell barrier within the nasal mucosal barrier.

[0021] This invention aims to provide an SBE-β-CD / F127 composite hydrogel drug delivery system by introducing SBE-β-CD (preferably with a mass fraction of 3% (w / w)) which has a supramolecular "molecular bridge" reinforcing effect. This system features a gelation temperature that can be precisely adjusted to 27.42379℃, a critical strain that can be increased to 0.1428, excellent operation performance at room temperature (25℃), instantaneous in-situ fixation performance in the nasal cavity at 35℃, high mechanical stability and strong resistance to dissolution, and ROS-triggered precise drug release characteristics.

[0022] This invention provides a cyclodextrin-bridged ROS-responsive thermosensitive gel and its applications. Through the microscopic topological enhancement and hydrogen bond network reconstruction of Pluronic F127 micelles using sulfobutyl ether-β-cyclodextrin (SBE-β-CD), a multifunctional drug delivery platform is constructed, possessing high mechanical strength, corrosion resistance, and oxidative stress-triggered release properties. This platform can serve as a non-invasive or minimally invasive sustained-release carrier for large molecule drugs such as proteins and peptides.

[0023] This invention provides an F127 / CD@TK-INS Gel composite hydrogel drug delivery system through three core technical measures: precise synthesis of ROS-responsive insulin prodrugs, optimized construction of SBE-β-CD / F127 thermosensitive composite gels, and deep coupling of prodrugs and gels. This system combines room-temperature injectable / sprayable operation, instantaneous in-situ gelation at 35°C in the nasal cavity, high mechanical stability and strong resistance to dissolution, and ROS-triggered precise drug release characteristics. The specific technical solution is as follows: A cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel, the gel comprising a thermosensitive polymer matrix, a functional modifier, and a ROS-responsive prodrug; The temperature-sensitive polymer matrix is ​​Pluronic F127; The functional modifier is sulfobutyl ether-β-cyclodextrin (SBE-β-CD), which acts as a "molecular bridge" to form an enhanced gel network with Pluronic F127 through hydrophobic inclusion and hydrogen bond reconstruction. ROS-responsive prodrugs are conjugates formed by functional groups linked by thioketal bonds and active drugs, and are uniformly dispersed in the enhanced gel network.

[0024] As a preferred embodiment of the above technical solution, the cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel provided by the present invention further includes some or all of the following technical features: As an improvement to the above technical solution, the mass fraction of the sulfobutyl ether-β-cyclodextrin (SBE-β-CD) is 0.5-5%, the mass fraction of the Pluronic F127 is 18-28%, the loading concentration of the ROS-responsive prodrug is 0.5-2 mg / mL, and the remainder is sterile water.

[0025] Blending ratio screening: The gelation behavior of different concentrations of F127 (18%-28%) and the addition of 3% SBE-β-CD was investigated, and 20% F127 + 3% SBE-β-CD was determined to be the optimal ratio. This system maintains good flowability, injectability and sprayability at 25°C, and the gelation rate is significantly faster than that of pure F127 at 35°C.

[0026] As an improvement to the above technical solution, the ROS-responsive prodrug is DHA-PEG. 2k -TK-Insulin, where DHA is docosahexaenoic acid, PEG is polyethylene glycol, TK is a thioketal bond, and Insulin is insulin.

[0027] As an improvement to the above technical solution, the sol-gel transition temperature of the composite hydrogel is 27.4±0.5℃, and the critical strain at 35℃ is greater than 0.14.

[0028] This invention also includes a method for preparing a cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel as described above, comprising the following steps: 1) Dissolve Pluronic F127 and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) in sterile water, stir and mix well, and let stand to obtain a composite gel matrix; specifically, add F127 and SBE-β-CD to the sterile aqueous phase, stir at 4°C until completely swollen, and let stand overnight to obtain a uniform and transparent thermosensitive composite gel matrix.

[0029] 2) After dissolving the ROS-responsive prodrug, add it to the composite gel matrix obtained in step 1) at a concentration of 1 mg / mL, and stir at 4°C until the prodrug is completely dispersed to obtain the hydrogel stock solution. The hydrogel mother liquor is a low-viscosity fluid at 25°C and is injectable / sprayable; it undergoes a rapid sol-gel transition at 35°C to form a three-dimensional interconnected porous network with a pore size of 0.8~1.2 μm.

[0030] As a preferred embodiment of the above technical solution, the method for preparing cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel as described above, provided by the present invention, further includes some or all of the following technical features: As an improvement to the above technical solution, the preparation method of the ROS-responsive prodrug is as follows: firstly, DHA-PEG is synthesized.2k -TK-NHS intermediates are then reacted with active drugs to obtain cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gels.

[0031] As an improvement to the above technical solution, the synthetic DHA-PEG 2k The method for obtaining the -TK-NHS intermediate is BOCNH-PEG. 2k -NH2 reacted with DHA at room temperature for 12 h under EDC / NHS catalysis. After acid washing, water washing, saturated brine washing, drying, and concentration, the product was deprotected with HCl / MeOH for 0.5 h, followed by alkali washing, water washing, saturated brine washing, drying, and concentration. The resulting product was reacted with NHS-TK-NHS at room temperature in the presence of TEA for 2 h. After recrystallization with acetone, solidification with ice-cold ether, and vacuum drying, DHA-PEG was obtained. 2k -TK-NHS (NMR verification of DHA connectivity rate 91.7%); the active drug in the ROS-responsive prodrug is a protein or peptide for treating neurodegenerative diseases.

[0032] As an improvement to the above technical solution, the protein-peptide drug is insulin, and the ROS-responsive prodrug is the ROS-responsive insulin prodrug DHA-PEG. 2k -TK-insulin.

[0033] As an improvement to the above technical solution, the ROS-responsive insulin prodrug DHA-PEG 2k The preparation method of -TK-insulin is to use DHA-PEG 2k The -TK-NHS intermediate was reacted with insulin at room temperature for 4 h in the presence of TEA. After precipitation with acetone, washing, and vacuum drying, the target prodrug was obtained. TLC dual colorimetric verification: the product showed color at Rf=0 under amino fluorescence and under PEG colorimetry, the product showed color at Rf=0 with no free carrier residue, proving that the coupling was successful.

[0034] The present invention also includes a pharmaceutical formulation comprising a ROS-responsive thermosensitive composite hydrogel reinforced with cyclodextrin molecular bridges as described in any of the preceding claims and a pharmaceutically acceptable carrier.

[0035] This invention also includes a pharmaceutical formulation comprising a ROS-responsive thermosensitive composite hydrogel reinforced with cyclodextrin molecular bridges as described in any of the preceding claims, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from one or more of the following: water, buffer solutions (e.g., phosphate buffer, acetate buffer, citrate buffer), isotonic adjusters (e.g., sodium chloride, glucose, mannitol), pH adjusters (e.g., hydrochloric acid, sodium hydroxide), preservatives (e.g., benzalkonium chloride, thimerosal, parabens), stabilizers (e.g., sucrose, trehalose, polyvinylpyrrolidone), thickeners (e.g., hydroxypropyl methylcellulose, carbomer), and surfactants (e.g., polysorbate 80, poloxamer 188). The pharmaceutical formulation can be prepared in various dosage forms, including but not limited to injections, sprays, nasal drops, implants, gels, lyophilized powder for injection, or in-situ gels. Based on experimental studies, the formulation preferably maintains fluidity at 25°C and rapidly forms a gel at 35°C, thus being suitable for nasal administration and possessing injectability and sprayability. The ROS-responsive prodrug can be DHA-PEG-TK-Insulin, or other suitable peptide or protein prodrugs, to achieve responsive release.

[0036] The core technical principles and features of this invention are described below: (1) Enhancement principle of SBE-β-CD supramolecular “molecular bridge” 3% SBE-β-CD selectively encapsulates the hydrophobic PPO segments of F127 through a hydrophobic cavity, simultaneously initiating the reconstruction of the PEO chain hydrogen bond network (infrared spectroscopy shows that the COC changes from 1085.65 cm⁻¹). - ¹Displaced to 1085.57 cm - ¹, OH is 3491.84 cm - ¹Displaced to 3496.46 cm - ¹), acting as a “molecular bridge” for F127 micelles, promotes higher-order micelle assembly. This mechanism achieves: Optimized gelation temperature: Reduced from 29.4005℃ ​​for pure F127 to 27.42379℃, which solves the problem of poor room temperature operability and adapts to the gelation requirements of 35℃ in the nasal cavity; Mechanical strength improvement: The critical strain increased from 0.1108 to 0.1428, and the complex shear modulus was significantly improved, breaking through the technical bottleneck of "heating and reducing strength" in traditional HP-β-CD.

[0037] (2) TK bond ROS response precise drug release principle DHA-PEG 2kThe TK bond in -TK-insulin is highly sensitive to ROS, stable in physiological environments, and specifically breaks in the 0.1 mM H2O2 microenvironment, reaching peak release within 8 hours, enabling on-demand dosing in response to the lesion microenvironment.

[0038] (3) The principle of dual synergistic controlled release of "physical interception-chemical triggering" Physical interception: The dense three-dimensional network of SBE-β-CD / F127 composite gel acts as a physical barrier, effectively delaying drug diffusion and solving the problem of nearly 100% drug release within 6 hours of pure F127 gel; Chemical triggering: The encapsulated prodrug achieves precise drug release through the ROS-responsive cleavage of the TK bond; Dual synergy: The deep coupling of the two enables the carrier to intelligently judge whether the physiological environment is stable or the lesion environment is suitable for drug release, while solving the common industry problems of "difficulty in mucosal retention" and "difficulty in precise release" in nasal drug delivery. Compared with the prior art, the technical solution of the present invention has the following beneficial effects: 1. Precisely matched gelation temperature to physiological window: Through the "molecular bridging" enhancement effect of 3% SBE-β-CD, the gel point is precisely adjusted from 29.4005℃ ​​of pure F127 to 27.42379℃, which not only ensures excellent fluidity at room temperature of 25℃ (injectable / sprayable), but also achieves rapid in-situ gelation at the physiological temperature of 35℃ in the nasal cavity, solving the problem of mismatch between the gelation temperature and the administration window of traditional F127.

[0039] 2. Significant improvement in mechanical strength and resistance to deformation: The critical strain of the composite gel increased from 0.1108 for pure F127 to 0.1428, and the complex shear modulus (G*) was significantly higher than that of pure F127 at all temperature points, resulting in a substantial increase in shear resistance. It can maintain structural integrity and is not easily disintegrated under physiological conditions such as the movement of nasal cilia.

[0040] 3. Significantly optimized resistance to dissolution and physical controlled release: Methylene blue release experiments showed that pure F127 gel released nearly 100% within 6 hours, while the release of composite gel was significantly delayed, proving that the dense three-dimensional network enhanced by SBE-β-CD can serve as an effective physical barrier and completely solve the problem of drug burst release.

[0041] 4. ROS-responsive drug release is precise and efficient: encapsulated DHA-PEG 2k -TK-insulin prodrug reaches peak DHA-PEG fragment release within 8 hours in a 0.1 mM H2O2 microenvironment, with a cumulative release significantly higher than that of the saline control group, achieving on-demand precise drug release in response to the disease microenvironment.

[0042] 5. Uniform and controllable microstructure: SEM shows that the composite gel forms a three-dimensional interconnected micron-sized porous network with a pore size mainly distributed at ~1μm. The uniform structure is conducive to the uniform loading and release of drugs, while providing a suitable space for the adhesion and penetration of nasal mucosal cells.

[0043] 6. Synergistic effect of dual controlled release mechanism: It combines the physical barrier controlled release of SBE-β-CD / F127 composite gel with chemically triggered drug release in response to TK bond ROS, realizing intelligent delivery with "stable physiological environment and drug release in lesion environment", while solving the common industry problems of "difficulty in mucosal retention" and "difficulty in precise release" in nasal drug delivery.

[0044] 7. Excellent biocompatibility and great potential for clinical translation: Both F127 and SBE-β-CD are FDA-approved pharmaceutical excipients. The prodrug synthesis conditions are mild and the preparation process is controllable, which gives them good prospects for clinical translation.

[0045] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, the following detailed description is provided in conjunction with preferred embodiments. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below.

[0047] Figure 1 It is DHA-PEG 2k Thin-layer chromatography (TLC) identification chromatogram of -TK-insulin; Figure 2 shows DHA-PEG. 2k -TK-insulin DHA-PEG release curve in physiological saline and 0.1 mM H2O2 medium; Figure 3 shows the state diagrams of F127 hydrogels with different concentrations at 25℃; Figure 4 These are state diagrams of F127+3%SBE-β-CD hydrogels at 25℃ with different concentrations. Figure 5 Thermosensitive phase transition behavior: Based on the supramolecular self-assembly of F127 / SBE-β-CD, it forms a sprayable sol at 25°C and rapidly gels at 35°C, thus prolonging nasal mucosal retention. Figure 6 Comparison of gelation time between F127 hydrogel (top) and F127+3% SBE-β-CD (bottom); Figure 7 shows the DLS hydration particle size distribution of F127 hydrogel; Figure 8 shows the DLS hydration particle size distribution of F127+3% SBE-β-CD hydrogel; Figure 9 shows the rheological properties of F127 hydrogel; Figure 10 This is a rheological property diagram of F127+3% SBE-β-CD hydrogel; Figure 11 These are SEM images of the F127 / CD@TK-INS Gel lyophilized hydrogel; Figure 12 shows the in-situ infrared spectra of F127 and F127+3% SBE-β-CD hydrogel; Figure 13 is a comparison of the methylene blue release curves of F127 hydrogel and F127+3% SBE-β-CD hydrogel; Figure 14 shows the sprayability of the 20% F127+3% SBE-β-CD hydrogel. Detailed Implementation

[0048] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can repeat the technical solution of the present invention according to the following steps. All process parameters and material ratios are the optimal values ​​verified by experiments. The operation is reproducible and the results are verifiable.

[0049] I. Experimental Reagents and Instruments (a) Experimental reagents BOCNH-PEG 2k -NH2, docosahexaenoic acid (DHA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), bissuccinimide ester thioacetate (NHS-TK-NHS), triethylamine (TEA), insulin, Pluronic F127, sulfobutyl ether-β-cyclodextrin (SBE-β-CD), methylene blue, dichloromethane (DCM), N,N-dimethylformamide (DMF), acetone, and anhydrous sodium sulfate were all of analytical grade; sterile deionized water, physiological saline, and 0.1 mM H2O2 solution were prepared fresh for use.

[0050] (II) Experimental Instruments Magnetic stirrer, rotary evaporator under reduced pressure, vacuum drying oven, scanning electron microscope (SEM), rheometer, in-situ infrared spectrometer, laser particle size analyzer (DLS), GF254 thin-layer chromatography plate, ultraviolet analyzer, constant temperature water bath, freeze dryer, 10mL centrifuge tubes, 50mL / 25mL round-bottom flasks. II. Specific Implementation Methods Example 1: ROS-responsive insulin prodrug (DHA-PEG) 2k Synthesis and Performance Verification of -TK-insulin This embodiment completes the DHA-PEG process. 2k The stepwise synthesis of the TK-insulin prodrug was performed. Figure 1 (TLC identification diagram) verified the coupling effect between insulin and the carrier, and Figure 2 (ROS response release curve) characterized the oxidative stress-specific drug release performance of the prodrug. The specific steps are as follows: 1.1 DHA-PEG 2k Synthesis of -TK-NHS intermediates Weigh out 300 mg of BOCNH-PEG 2k -NH2 was placed in a 50 mL round-bottom flask, and 20 mL of DCM was added and sonicated until completely dissolved. Then, 1.0 eq DHA, 3.0 eq EDC, and 3.0 eq NHS were added sequentially, and the mixture was reacted at room temperature for 12 h under magnetic stirring. After the reaction was completed, the mixture was concentrated to 5 mL under reduced pressure at 40 °C, and 10 mL of DCM was added to redissolve it. The mixture was washed three times with acid water, twice with sterile deionized water, and once with saturated saline solution. The organic phase was dried with anhydrous sodium sulfate for 2 h, filtered, and concentrated again under reduced pressure at 40 °C.

[0052] HCl / MeOH (volume ratio 1:9) was added to the above concentrated product, and the product was deprotected at room temperature for 0.5 h. The solvent was removed by concentration under reduced pressure, and 10 mL of DCM was added for redissolution. The product was washed twice with alkaline water, twice with sterile deionized water, and once with saturated saline. After drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, the product was slowly added dropwise to 20 mL of DCM solution containing 5.0 eq NHS-TK-NHS and 3.0 eq TEA. The mixture was stirred at room temperature for 2 h. After concentration under reduced pressure, 10 mL of acetone was added for recrystallization. After filtration, the solid was solidified three times with ice-cold ether and dried under vacuum for 24 h to obtain a white solid powder DHA-PEG. 2k -TK-NHS, as detected by 1H NMR spectroscopy, showed a DHA connectivity rate of 91.7%.

[0053] 1.2 DHA-PEG 2k Synthesis of TK-insulin prodrug Weigh 75 mg of insulin into a 25 mL round-bottom flask, add sterile deionized water and sonicate to dissolve. Add 3.0 eq DHA-PEG. 2k5 mL of DMF solution and 3.0 eq TEA of -TK-NHS were added and reacted at room temperature for 4 h with magnetic stirring. After the reaction, the DMF was removed by concentration under reduced pressure at 40 °C. 20 mL of acetone was added to the remaining aqueous solution to precipitate the product. After standing for 30 min, the mixture was filtered. The solid was washed twice with acetone and dried under vacuum for 24 h to obtain a white solid powder, DHA-PEG. 2k -TK-insulin.

[0054] 1.3 Verification of Coupling Effect in TLC (corresponding to Figure 1) Figure 1 It is DHA-PEG 2k Thin-layer chromatography (TLC) identification diagram of -TK-insulin (left: amino fluorescence; right: PEG), labeled A (DHA-PEG). 2k -TK-NHS), B (insulin), C (DHA-PEG) 2k (-TK-insulin), to verify the efficient coupling of insulin with the carrier and the absence of free carrier residue.

[0055] Using GF254 silica gel plates and a methanol:water ratio of 8:2 as the developing solvent, samples A (DHA-PEG) were spotted separately. 2k -TK-NHS), B (insulin), C (DHA-PEG) 2k -TK-insulin), verified by double colorimetric method: Amino fluorescent color development: Spray amino fluorescent color developer onto the silica gel plate and observe under ultraviolet light. No fluorescence is observed at point A, while strong fluorescence is observed at Rf=0 at points B and C, proving that the product at point C successfully binds to amino-containing insulin. PEG color development: PEG color developer was sprayed onto a silica gel plate and heated. Point A showed color development at Rf=0.6, point B showed no color development, and point C showed significant color development at Rf=0 with no residue at Rf=0.6, demonstrating the interaction between insulin and DHA-PEG. 2k -TK-NHS is fully coupled and free of free carrier impurities.

[0056] 1.4 ROS-responsive drug release performance verification (corresponding to Figure 2) Prepare 0.5 mg / mL DHA-PEG 2k-TK-insulin aqueous solution was divided into two groups: the control group was given an equal volume of physiological saline, and the experimental group was given an equal volume of 0.1 mM H2O2 solution. Both groups were placed in a constant temperature water bath at 37℃ and shaken. Samples were taken at 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h to detect the release rate of DHA-PEG fragments. The results showed that the release rate of the experimental group reached its peak within 8 hours, and the cumulative release was significantly higher than that of the physiological saline control group, proving that the prodrug has a highly efficient and specific response to the ROS microenvironment.

[0057] Example 2: Preparation and performance characterization of SBE-β-CD / F127 thermosensitive composite gel Figure 2 shows DHA-PEG. 2k The DHA-PEG release curve of TK-insulin in physiological saline and 0.1 mM H2O2 medium, with the horizontal axis representing time and the vertical axis representing release rate (%), characterizes the ROS-specific response release behavior of the prodrug.

[0058] This embodiment completes the gradient screening of F127 single component and compound system, determines the optimal ratio, and uses Figure 3 and... Figure 4 (Figure 3 shows the state diagrams of F127 hydrogels at different concentrations (18%, 20%, 22%, 24%, 26%, 28%) at 25℃; Figure 4 shows the state diagrams of F127+3%SBE-β-CD hydrogels at different concentrations (18%, 20%, 22%, 24%, 26%, 28%) at 25℃.) Characterizing the gelation boundary of a single component, Figure 5 and... Figure 6 (Figure 5 and) Figure 6 This is a comparison of the gelation states of 20% F127 + 3% SBE-β-CD hydrogel at 25℃ and 35℃, visually demonstrating the thermosensitive characteristics of the compound system's room temperature flow and rapid gelation at nasal cavity temperature. (Verification of thermosensitive gelation performance, Figure 7 and...) Figure 8 (Figure 7 shows the DLS hydration particle size distribution of F127 hydrogel, with the horizontal axis representing particle size (d.nm) and the vertical axis representing abundance, characterizing the higher-order assembly regulation and "molecular bridge" enhancement effect of SBE-β-CD on F127 micelles; Figure 8 shows the DLS hydration particle size distribution of F127+3% SBE-β-CD hydrogel, with the horizontal axis representing particle size (d.nm) and the vertical axis representing abundance, characterizing the higher-order assembly regulation and "molecular bridge" enhancement effect of SBE-β-CD on F127 micelles.) Analysis of micelle assembly state, Figure 9 and... Figure 10(Figure 9 shows the rheological properties of F127 hydrogel, and Figure 10 shows the rheological properties of F127+3% SBE-β-CD hydrogel. The curves showing the changes in G' / G'' with temperature, strain amplitude, and frequency, and the curves showing the changes in G* with temperature, characterize the significant improvement in the mechanical strength and deformation resistance of the composite gel.) Mechanical strength was tested, and Figure 13 (a comparison of the methylene blue release curves of F127 hydrogel and F127+3% SBE-β-CD hydrogel, with the horizontal axis representing time and the vertical axis representing the release rate (%), characterizing the dense network physical barrier effect and controlled-release capability of the composite gel) was used to verify the physical controlled-release capability. The specific steps are as follows: 2.1 Preparation and flowability testing of F127 hydrogels with different concentrations (corresponding to Figure 3 and Figure 4) Figure 4 ) Weigh 1.8 g, 2.0 g, 2.2 g, 2.4 g, 2.6 g, and 2.8 g of F127 respectively into 10 mL centrifuge tubes, add sterile deionized water to a final volume of 10 mL, and stir magnetically at 4°C until completely dissolved to obtain F127 aqueous solutions with mass fractions of 18%, 20%, 22%, 24%, 26%, and 28% (w / w). The fluidity was observed after standing at 25°C for 30 min. The results showed that 18%, 20%, and 22% F127 maintained good fluidity (flowing freely when inverted), while concentrations of 24% and above completely gelled (no flow when inverted), thus determining the room temperature gelation concentration boundary for F127.

[0059] 2.2 Preparation and Optimal Ratio Screening of F127 / SBE-β-CD Compound Gel (corresponding to Figure 5 and...) Figure 6 ) 0.3 g of SBE-β-CD was added to each of the above-mentioned F127 aqueous solutions of different concentrations, and the mixture was magnetically stirred at 4℃ until completely dissolved. The volume was then adjusted to 10 mL to obtain a 18%~28% F127+3% SBE-β-CD compound gel system. The gelation properties were observed after standing at 25℃ for 30 min. The results showed that the compound system with a concentration of 22% and above gelled at 25℃. The 20% F127+3% SBE-β-CD compound gel maintained good fluidity and rapidly gelled within 15 s in a constant temperature water bath at 35℃. It could also be smoothly injected through a syringe and form uniform droplets through a sprayer, which was determined to be the optimal compound ratio.

[0060] 2.3 Verification of the gelation properties of high-proportion SBE-β-CD Weigh 2.0 g of F127 and 2.0 g of SBE-β-CD, add sterile deionized water to a final volume of 10 mL, and stir at 4℃ to dissolve, obtaining a 20% F127 + 20% SBE-β-CD compound system. The system was then allowed to stand for 30 min at constant temperatures of 25℃, 35℃, and 45℃. The results showed that the system remained liquid at all three temperatures without gelation, proving that high concentrations of SBE-β-CD would impair the micelle-forming ability of F127, and therefore it can only be used as a modifier at low concentrations.

[0061] 2.4 DLS detection of micelle assembly state (corresponding to Figure 7 and...) Figure 8 ) 20% F127 gel and 20% F127+3% SBE-β-CD composite gel were taken respectively, and the hydrated particle size of DLS was detected by laser particle size analyzer. The results showed that pure F127 showed two characteristic peaks at 4.19 nm and 50.7 nm, while the composite gel showed three characteristic peaks at 3.12 nm, 13.5 nm and 58.8 nm. This proves that SBE-β-CD, as a supramolecular "molecular bridge", can promote the higher-order assembly of F127 micelles, change the micelle size distribution, and strengthen the gel network structure.

[0062] 2.5 Rheological property testing (corresponding to Figure 9 and...) Figure 10 ) Multidimensional scanning was performed on 20% F127 gel and 20% F127+3% SBE-β-CD compound gel using a rheometer under fixed parameters. The results are as follows: Temperature scan (frequency 1 Hz, strain 1%): The gelation temperature of pure F127 gel is 29.4005℃, while the gelation temperature of the compound gel is precisely reduced to 27.42379℃. Moreover, the complex shear modulus (G*) of the compound gel is significantly higher than that of pure F127 at all temperature points. Strain amplitude scanning (temperature 35℃, frequency 1 Hz): the critical strain of pure F127 gel is 0.1108, while the critical strain of the compound gel is increased to 0.1428, and the resistance to deformation is significantly enhanced. Frequency scanning (temperature 35℃, strain 1%): The compound gel exhibits stable mechanical properties with no frequency dependence and good solid gel behavior over a wide frequency range of 0.01~100 Hz, with the storage modulus (G') always greater than the loss modulus (G'').

[0063] 2.6 Physical controlled release capability test (corresponding to Figure 13) Methylene blue was used as a model drug and encapsulated at a concentration of 1 mg / mL in 20% F127 gel and 20% F127+3% SBE-β-CD compound gel, respectively. The drug-loaded gels were placed in excess sterile deionized water and subjected to in vitro release experiments with constant temperature shaking at 37℃. Methylene blue release rate was measured at regular intervals. The results showed that the release rate of pure F127 gel was close to 100% within 6 hours, indicating a serious burst release phenomenon. The release rate of the compound gel was significantly delayed, proving that its dense three-dimensional network structure can form an effective physical barrier, solve the problem of drug burst release, and achieve long-acting sustained and controlled release.

[0064] Example 3: Preparation and microstructure and molecular interaction characterization of F127 / CD@TK-INS Gel composite hydrogel This embodiment completes the co-assembly of ROS-responsive prodrug and thermosensitive composite gel to prepare F127 / CD@TK-INSGel composite hydrogel. The microstructure of the gel is characterized by the SEM image (scale bar 1 μm) of the F127 / CD@TK-INSGel lyophilized hydrogel in Figure 11, which shows the three-dimensional interconnected micron-sized porous network microstructure of the composite hydrogel. Figure 12 (Figure 12 shows the in-situ infrared spectra of F127 and F127+3% SBE-β-CD hydrogels, with the horizontal axis representing wavenumber (cm).) - ¹), with the ordinate representing transmittance (%), characterizing the peak shifts of COC and OH, demonstrating the hydrophobic inclusion and hydrogen bond network reconstruction between SBE-β-CD and F127. The molecular interaction mechanism between SBE-β-CD and F127 is verified through the following steps: 3.1 Preparation of composite hydrogels Weigh 2.0 g of F127 and 0.3 g of SBE-β-CD, add 8.7 mL of sterile deionized water, and stir magnetically at 4°C until completely dissolved to obtain a 20% F127 + 3% SBE-β-CD composite gel matrix; weigh 10 mg of the DHA-PEG prepared in Example 1. 2k Dissolve TK-insulin in 1.0 mL of sterile deionized water, and slowly add it dropwise to the above composite gel matrix. Stir magnetically at 4°C for 30 min until the prodrug is uniformly dispersed to obtain a 1 mg / mL F127 / CD@TK-INS Gel composite hydrogel stock solution. This stock solution is a low-viscosity fluid at 25°C, with excellent injectability and sprayability, and rapidly gels within 15 s at 35°C.

[0065] 3.2 Microscopic morphology SEM characterization (corresponding to Figure 11) The above-mentioned composite hydrogel stock solution was placed in a constant temperature incubator at 37℃ for 10 min to form a complete gel. After being rapidly cooled and fixed with liquid nitrogen and freeze-dried, the microstructure was observed using SEM (scale bar 1 μm). The results showed that the composite hydrogel exhibited a three-dimensional interconnected micron-sized porous network structure with regular circular / elliptical pores and a uniform pore size distribution (mainly about 1 μm). The pores were connected by narrow channels, and the gel framework was a continuous polymer network, which was suitable for uniform drug loading and adhesion to the nasal mucosa.

[0066] 3.3 In-situ infrared verification of molecular interactions (corresponding to Figure 12) In situ infrared spectroscopy was used at 37℃ to detect the infrared absorption spectra of 20% F127 gel and 20% F127+3% SBE-β-CD composite gel, respectively; the results showed: The compound gel at 1100 cm - ¹ A peak of 0.08 cm⁻¹ appears near the F127 ether bond COC stretching vibration. - ¹ Displacement (from 1085.65 cm) - ¹ Up to 1085.57 cm - ¹), the peak height decreased, proving that the hydrophobic cavity of SBE-β-CD can selectively capture the PPO hydrophobic segment of F127; The compound gel at 3400 cm - ¹ A significant shift occurs near the hydroxyl (OH) stretching vibration peak (from 3491.84 cm⁻¹). - ¹ Up to 3496.46 cm - ¹), demonstrating that the interaction between SBE-β-CD and F127 triggers the reconstruction of the hydrogen bond network in the system, further stabilizing the three-dimensional network of the gel.

[0067] Example 4: Validation of the synergistic drug release performance of F127 / CD@TK-INS Gel composite hydrogel This embodiment, in conjunction with Figure 2 (ROS release curve) and Figure 13 (methylene blue release curve), verifies the dual synergistic effect of the composite hydrogel's "physical barrier sustained-release + ROS chemically triggered drug release." The specific steps are as follows: The F127 / CD@TK-INS Gel composite hydrogel stock solution was divided into two groups and placed in a 37℃ constant temperature water bath for in vitro drug release experiment: the control group was added with an equal volume of physiological saline, and the experimental group was added with an equal volume of 0.1 mM H2O2 solution. Samples were taken at regular intervals to detect the insulin release rate. The results showed that in the control group, insulin was released slowly and at a low rate due to the physical barrier effect of the dense three-dimensional network of the gel, without any burst release. In the experimental group, the TK bonds in the prodrug were specifically broken under the oxidative stress of H2O2, triggering insulin release. At the same time, the physical barrier effect of the gel effectively delayed drug diffusion and avoided burst release. Ultimately, the synergistic effect of "physical interception-chemical triggering" dual controlled release was achieved, making insulin exhibit slow, continuous, and lesion-specific release characteristics, which can meet the needs of precise and long-term delivery of protein and peptide drugs through non-invasive nasal administration.

[0068] Example 5: Validation of the room temperature injectability and sprayability of the 20% F127+3% SBE-β-CD composite hydrogel The 20% F127+3% SBE-β-CD composite hydrogel (loaded with DHA-PEG-TK-insulin 1mg / mL) prepared in Example 3 was equilibrated at 25℃ for 30 minutes, and its injectability and sprayability were tested respectively.

[0069] Injectability test: The hydrogel was loaded into a 5 mL medical syringe (with a 21G needle) and manually injected. Results are as follows: Figure 5 As shown, the hydrogel was smoothly absorbed and injected continuously in a line without clogging, phase separation, or needle blockage. The extrudate was uniform and transparent, demonstrating that the system has excellent injectability at room temperature.

[0070] Sprayability test: Hydrogel was loaded into a commercially available nasal spray bottle and manually sprayed. Results are as follows: Figure 14 As shown in Figure 14, the sprayability of the 20% F127+3% SBE-β-CD hydrogel is demonstrated. The excellent handling performance of the compound system at room temperature was characterized by a sprayer. When pressed, the hydrogel is uniformly atomized and sprayed out with fine and uniform droplets. The spray is continuous and smooth, without clogging, dripping, or stringing, proving that the system also has good sprayability at room temperature.

[0071] The above results indicate that the hydrogel has both injectable and aerosol-like properties at room temperature, making it easy to load into syringes or spray devices before clinical use, thus providing a convenient operating window for nasal drug delivery.

[0072] All the raw materials listed in this invention, as well as the upper and lower limits and ranges of the raw materials and the upper and lower limits and ranges of the process parameters (such as temperature, time, etc.), can realize this invention. Examples are not listed one by one here.

[0073] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel, characterized in that: The gel comprises a thermosensitive polymer matrix, a functional modifier, and a ROS-responsive prodrug; The temperature-sensitive polymer matrix is ​​Pluronic F127; The functional modifier is sulfobutyl ether-β-cyclodextrin (SBE-β-CD), which acts as a "molecular bridge" to form an enhanced gel network with Pluronic F127 through hydrophobic inclusion and hydrogen bond reconstruction. ROS-responsive prodrugs are conjugates formed by functional groups linked by thioketal bonds and active drugs, and are uniformly dispersed in the enhanced gel network. The mass fraction of the sulfobutyl ether-β-cyclodextrin (SBE-β-CD) is 3%, the mass fraction of the Pluronic F127 is 18-22%, the loading concentration of the ROS-responsive prodrug is 0.5-2 mg / mL, and the remainder is sterile water; The ROS-responsive prodrug is DHA-PEG. 2k -TK-Insulin, where DHA is docosahexaenoic acid, PEG is polyethylene glycol, TK is a thioketal bond, and Insulin is insulin; The sol-gel transition temperature of the cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel is 27.4±0.5℃, and the critical strain at 35℃ is greater than 0.

14.

2. A method for preparing a cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel as described in claim 1, characterized in that, It includes the following steps: 1) Dissolve Pluronic F127 and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) in sterile water, stir and mix well, and let stand to obtain a composite gel matrix; 2) After dissolving the ROS-responsive prodrug, add it to the composite gel matrix obtained in step 1) and mix evenly to obtain the hydrogel mother liquor; the hydrogel mother liquor is a low-viscosity fluid at 25°C and has injectable / sprayable properties; it undergoes a rapid sol-gel transition at 35°C to form a three-dimensional interconnected porous network with a pore size of 0.8~1.2 μm. The preparation method of the ROS-responsive prodrug is as follows: firstly, DHA-PEG is synthesized. 2k -TK-NHS intermediates are then reacted with active drugs to obtain cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gels.

3. The method for preparing cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel as described in claim 2, characterized in that: The synthetic DHA-PEG 2k The method for obtaining the -TK-NHS intermediate is BOCNH-PEG. 2k -NH2 reacted with DHA at room temperature for 12 h under EDC / NHS catalysis. After acid washing, water washing, saturated brine washing, drying, and concentration, the product was deprotected with HCl / MeOH for 0.5 h, followed by alkali washing, water washing, saturated brine washing, drying, and concentration. The resulting product was reacted with NHS-TK-NHS at room temperature in the presence of TEA for 2 h. After recrystallization with acetone, solidification with ice-cold ether, and vacuum drying, DHA-PEG was obtained. 2k -TK-NHS; The active drug in the ROS-responsive prodrug is a protein or polypeptide used to treat neurodegenerative diseases; The protein-peptide drug is insulin, and the ROS-responsive prodrug is the ROS-responsive insulin prodrug DHA-PEG. 2k -TK-insulin.

4. The method for preparing cyclodextrin molecular bridge-enhanced ROS-responsive thermosensitive gel as described in claim 3, characterized in that: The ROS-responsive insulin prodrug DHA-PEG 2k The preparation method of -TK-insulin is to use DHA-PEG 2k -TK-NHS intermediate reacted with insulin at room temperature for 4 h in the presence of TEA, and after precipitation with acetone, washing and vacuum drying, the target prodrug was obtained; TLC dual colorimetric verification: the product showed color at Rf=0 under amino fluorescence colorimetry, and the product showed color at Rf=0 under PEG colorimetry with no free carrier residue, proving that the coupling was successful.

5. A pharmaceutical preparation, characterized in that: The product comprises a ROS-responsive thermosensitive composite hydrogel reinforced with cyclodextrin molecular bridges as described in claim 1 and a pharmaceutically acceptable carrier.

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