Preparation method of a dual-response thermosensitive gel drug carrier, preparation and application thereof

Abstract

This invention relates to a method for preparing a dual-responsive thermosensitive gel drug carrier, belonging to the field of biopharmaceutical technology. The specific steps of the method for preparing the dual-responsive thermosensitive gel for drug carrier are as follows: ROS-sensitive thioketal bonds and hyaluronic acid are co-modified with amino and azido groups on MSN, respectively, coupled with mPEG-NH2, and then constructed using an inulin / poloxam composite thermosensitive gel semi-interpenetrating system to form the dual-responsive thermosensitive gel for drug carrier. Curcumin is further prepared by loading curcumin onto azidoamine-aminated mesoporous silica nanoparticles. This can bypass the blood-brain barrier via the nasobrain pathway, target inflammatory activated cells, and responsively cleave in a ROS-rich environment to release the drug, increasing the drug concentration at the lesion site and improving the drug's bioavailability in the brain. This achieves synergistic treatment of Alzheimer's disease through the gel and targeted responsive drug delivery, providing a more suitable and effective new drug delivery method.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a method for preparing a dual-response thermosensitive gel drug carrier, its formulation, and its application. Background Technology

[0002] Alzheimer's disease (AD) is an insidious neurodegenerative disease of the central nervous system. Current research generally suggests that the pathogenesis of AD is not driven by a single factor, but rather by a vicious cycle of oxidative stress and neuroinflammation. Excessive reactive oxygen species (ROS) induce mitochondrial dysfunction and neuronal damage, while the deposition of toxic β-amyloid protein (Aβ) continuously activates microglia, releasing large amounts of inflammatory factors, further exacerbating oxidative damage to the brain microenvironment. The blood-brain barrier (BBB) ​​constitutes a natural anatomical barrier. Through its tightly packed, low-permeability structure and the active pumping mechanism of efflux transporters such as P-glycoproteins, it keeps most small-molecule drugs and almost all large-molecule preparations out of brain tissue, resulting in brain drug concentrations far below the effective therapeutic threshold. This is the core bottleneck currently facing AD drug development.

[0003] To bypass the blood-brain barrier, the nose-to-brain drug delivery strategy has received widespread attention in recent years. After absorption through the nasal mucosa, drugs can directly enter the brain via the olfactory and trigeminal nerve pathways, achieving non-invasive and rapid central drug delivery. However, this pathway also faces significant challenges: the cilia covering the nasal mucosa have a rapid clearance function, causing the instilled drug solution to be physiologically flushed into the pharynx within minutes to tens of minutes, resulting in a very short residence time of the drug within the absorption window and severely limiting bioavailability.

[0004] To improve drug retention time in the nasal cavity, in-situ thermosensitive hydrogels have become an ideal carrier strategy. Poloxamer is the most widely used thermosensitive gel matrix, possessing excellent properties of being a free-flowing liquid at room temperature and rapidly gelling at body temperature, facilitating nasal instillation. However, poloxamer inherently has weak bioadhesion, and relying solely on its thermosensitive gelation cannot overcome the strong clearance by mucociliary cells.

[0005] Regarding the selection of active pharmaceutical ingredients, curcumin possesses multi-target pharmacological activity, capable of intervening in the progression of Alzheimer's disease (AD) through multiple pathways, including inhibiting Aβ aggregation, regulating the NF-κB pathway to antagonize neuroinflammation, and scavenging free radicals to reduce oxidative stress. However, curcumin itself suffers from fatal drawbacks such as extremely poor water solubility, rapid in vivo metabolism, and insufficient absolute oral bioavailability. Although there have been reports of encapsulating curcumin in nanodelivery systems, existing formulations generally lack lesion microenvironment-responsive drug release capabilities, leading to premature drug leakage into the bloodstream or non-lesion sites. This not only reduces efficacy but may also cause off-target toxicity.

[0006] Notably, the microenvironment of Alzheimer's disease (AD) lesions exhibits high concentrations of reactive oxygen species (ROS), providing an endogenous triggering condition for designing "smart-gated" release strategies. Thioketal (TK) bonds are typical ROS-responsive groups, structurally stable in low-ROS physiological environments but capable of specific breakage in high-ROS pathological microenvironments. They have been used as molecular switches for targeted drug release from tumor and inflammatory sites. Simultaneously, in inflammatory brain states, activated microglia significantly overexpress CD44 receptors, and hyaluronic acid (HA), as a specific ligand for CD44, can mediate active targeted endocytosis of nanoparticles through receptor-ligand recognition. However, there are currently no reports of co-modifying the active inflammatory targeting of HA with the microenvironment-responsive breakage of TK in the same delivery system.

[0007] In summary, the existing technology faces the following pressing technical challenges that need to be addressed:

[0008] First, there is a lack of curcumin delivery platforms that can simultaneously achieve long-term retention in the nasal mucosa, actively target inflammatory cells in the brain, and release drugs on demand in response to the ROS concentration in the AD microenvironment.

[0009] Second, gating and targeting functions are difficult to integrate synergistically. Existing MSN modification schemes are mostly single-function modifications, or although multiple functions are introduced, the modification steps are complicated and functional groups interfere with each other, making it difficult to achieve efficient synergistic integration of "gating and controlled release" and "active targeting" functions on the same nanoparticle.

[0010] Third, the problem of poor adhesion performance of poloxamer thermosensitive hydrogel has not been fundamentally improved. Existing inulin composite strategies only remain at the level of simple physical mixing and have not functionalized and synergistically designed their molecular adhesion mechanism and thermosensitive phase change characteristics.

[0011] Fourth, the brain-targeted drug delivery system, which integrates HA-mediated inflammatory cell targeting, TK bond-mediated ROS-responsive drug release, inulin-enhanced mucosal adhesion, and poloxamer's thermosensitive gelling properties, has been constructed with a three-tiered progressive function: "nasal mucosal adhesion anchoring - active recognition of inflammatory cells - ROS-triggered in-situ explosive release." To date, no technology or preparation reports have been found.

[0012] To date, existing technologies have neither recognized the physicochemical compatibility barriers of the above combination, nor have they proposed any complete solution that can synergistically achieve long-term nasal retention, active recognition of inflammatory cells, intelligent drug release in the ROS microenvironment, and high drug loading stability. Summary of the Invention

[0013] In view of this, the purpose of this invention is to provide a method for preparing a dual-response thermosensitive gel for drug carriers, and the obtained dual-response thermosensitive gel, as well as a method for preparing a curcumin-dual-response thermosensitive gel. This invention first prepares mesoporous silica nanoparticles orthogonally modified with amino and azido groups. By simultaneously introducing amino and azido groups onto the MSN surface, a ROS-responsive thioacetate-mPEG-gated molecule is linked via an amidation reaction of the amino group with NHS ester. Simultaneously, a copper-free click chemistry reaction of the azido group with dibenzocyclooctylene (DBCO) is used to link a hyaluronic acid targeting ligand. The two coupling reactions do not interfere with each other and are sequentially controllable, achieving a synergistic integration of gating and targeting functions. Furthermore, the nanoparticles are combined with inulin, ... Poloxamer, through a cryo-concentration effect induced by ice crystal growth, drives the formation of an irreversible microcrystalline physical cross-linked network of P407's PEO blocks. This network, combined with the subsequent formation of a reversible PPO micelle network upon heating, forms a dual framework, thereby topologically locking the interspersed inulin macromolecules and creating a composite thermosensitive gel semi-interpenetrating system. This system facilitates easy nasal spraying and long-lasting adhesion within the nasal cavity, enabling drug delivery via the naso-brain pathway. It forms an integrated naso-brain targeted drug delivery system characterized by nasal mucosal adhesion and anchoring, active recognition by inflammatory cells, and ROS-triggered in-situ explosive release. This provides a novel technical solution for constructing intelligent nanomedicine delivery systems.

[0014] To achieve the above objectives, the present invention specifically provides the following technical solution:

[0015] A first aspect of the present invention is to provide a method for preparing a dual-response thermosensitive gel for a drug carrier, the method comprising the following specific steps:

[0016] Preparation of S1, N3 / NH2-MSN:

[0017] MSNs were added to xylene containing a mixture of APTES and AzPTES. After stirring thoroughly under nitrogen at room temperature for 24 hours, the mixture was centrifuged, washed three times with anhydrous ethanol, and then dried under vacuum at 50°C for 12 hours to obtain a white powder product, which is N3 / NH2-MSN.

[0018] Preparation of S2 and TK-MSN:

[0019] 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, EDC·HCl, and NHS were added to MES buffer solution and stirred at room temperature under nitrogen atmosphere in the dark to carry out an acylation reaction, yielding the ROS-responsive crosslinking agent TKDA. Then, a dispersed N3 / NH2-MSN solution was added dropwise, and the mixture was stirred at room temperature under nitrogen atmosphere in the dark for 24–48 hours. The precipitate was collected by centrifugation and washed with acetone and ultrapure water. After washing, the product was vacuum dried at room temperature to obtain a white precipitate, TK-MSN.

[0020] Preparation of S3 and PEG-TK-MSN:

[0021] EDC·HCl and NHS were added to the dimethyl sulfoxide solution of TK-MSN and the reaction was activated for 1.5 to 3 hours. The solution was then slowly added dropwise to a 10 to 20 mg / mL mPEG-NH2 solution and stirred at room temperature under nitrogen cover in the dark for 24 to 36 hours. The precipitate was collected by centrifugation and washed alternately with anhydrous ethanol and acetone. The precipitate was then freeze-dried at -50°C under vacuum to obtain the final product PEG-TK-MSN.

[0022] Preparation of S4, PEG-HA & TK-MSN:

[0023] EDC·HCl, NHS, and HA were dissolved in MES buffer and activated by gentle stirring at room temperature in the dark for 20-30 min. DBCO-NH2 was then added, and the pH was adjusted to approximately 7.4 with NaOH. The mixture was stirred overnight at room temperature to synthesize HA-DBCO. The reaction solution was placed in a dialysis bag (MWCO 10 kDa) and dialyzed in ultrapure water for 48 h. The solution was then lyophilized to obtain dry HA-DBCO, which was added to a PEG-TK-MSN suspension. The reaction was stirred under nitrogen atmosphere at room temperature in the dark for 24-36 h. The product was collected by centrifugation, washed 3-5 times with ultrapure water, and then freeze-dried at -50℃ under vacuum to obtain PEG-HA&TK-MSN.

[0024] S5. Preparation of thermosensitive gel:

[0025] Add poloxamer P407, poloxamer P188, and inulin to a 10-20 mg / mL PEG-HA&TK-MSN solution. Stir at room temperature until the poloxamer and inulin dissolve. Let the mixture stand at 3-8℃ for 18-24 hours, then freeze at -20℃ for 2 hours and alternate between freeze-thaw at room temperature. Repeat this freeze-thaw cycle at least 3 times before thawing at room temperature to obtain the dual-response thermosensitive gel.

[0026] This invention innovatively incorporates inulin into the poloxamer system. Utilizing the freeze-concentration effect induced by ice crystal growth, it drives the PEO blocks of P407 to form an irreversible microcrystalline physical cross-linked network. This network, together with the reversible PPO micelle network formed by subsequent heating, constitutes a dual framework, thereby topologically locking the interspersed inulin macromolecules and forming a physically semi-interpenetrating structure. This not only thickens the system and enhances its stability in a temperature-sensitive gel system but also helps improve the dispersion stability of drug-loaded nanoparticles within the gel, preventing their aggregation and precipitation. Existing technologies simply mix inulin into the gel without synergistically constructing a drug delivery system with other functional responsive groups.

[0027] Furthermore, in the preparation method of the dual-response thermosensitive gel, the molar ratio of azido groups and amino groups modified on N3 / NH2-MSN in step S1 is 10~12:3~5.

[0028] Furthermore, in the preparation method of the dual-response thermosensitive gel, the molar ratio of 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, EDC·HCl and NHS in step S2 is 1:1.5~2:1~2.

[0029] Furthermore, in the preparation method of the dual-response thermosensitive gel, the acylation reaction in step S2 lasts for 0.5 to 3 hours.

[0030] Furthermore, in the preparation method of the dual-response thermosensitive gel, the mass concentration of N3 / NH2-MSN in step S2 is 5-10 mg / mL.

[0031] Furthermore, in the preparation method of the dual-response thermosensitive gel, the mass concentration of TK-MSN in step S3 is 8-15 mg / mL.

[0032] Furthermore, in the preparation method of the dual-response thermosensitive gel, the mass ratio of HA&TK-MSN, EDC·HCl, NHS and mPEG-NH2 in step S3 is 1:0.1~0.5:0.1~0.5:1~1.5.

[0033] Furthermore, in the preparation method of the dual-response thermosensitive gel, the mass ratio of EDC·HCl, NHS and HA in step S4 is 1.5~2:0.8~1.2:1.

[0034] Furthermore, in the preparation method of the dual-response thermosensitive gel, the mass ratio of PEG-HA&TK-MSN, poloxamer P407, poloxamer P188 and inulin in step S5 is 1~2:18~22:2~4:2.

[0035] Furthermore, in the preparation method of the dual-response thermosensitive gel, the MSNs are prepared by adding PEG300 and NaOH to CTAB and stirring at 75℃~80℃ for 30min~60min; then slowly adding TEOS and continuing to stir for 1~2h. After centrifugation, the gel is washed three times with ultrapure water and ethanol respectively, and then vacuum dried to obtain a white precipitate. In order to obtain ordered mesoporous channels, the template is removed by using an acidic methanol solution. That is, 1g of white precipitate is placed in 169mL of acidic methanol solution (160mL methanol, 9mL hydrochloric acid), sonicated to disperse it fully, and then stirred and heated at 60℃ for 12h-14h to remove CTAB. After centrifugation again, the gel is washed 3-5 times with ultrapure water and ethanol respectively, and then vacuum dried at 50℃ for 12h to obtain MSNs.

[0036] Furthermore, 0.5g MSNs were added to a mixture of 0.234mL APTES and 0.126mL A Z The white powder product obtained by stirring PTES in 50 mL of xylene under nitrogen at room temperature for 24 h, followed by centrifugation, washing three times with anhydrous ethanol, and vacuum drying at 50 °C for 12 h is N3 / NH2-MSN.

[0037] A second aspect of the present invention is to provide a dual-response thermosensitive gel for use as a drug carrier, obtained by the method for preparing the dual-response thermosensitive gel provided in the first aspect above.

[0038] The third aspect of the present invention is to provide a method for preparing curcumin-dual-response thermosensitive gel by the method for preparing dual-response thermosensitive gel provided in the first aspect above. Specifically, in step S1, N3 / NH2-MSN is replaced with N3 / NH2-MSN / Cur. The preparation method of N3 / NH2-MSN / Cur is as follows: N3 / NH2-MSN is added to an anhydrous ethanol solution of curcumin with a concentration of 4~10 mg / mL, pH 6.5~pH 7, stirred at room temperature in the dark for 24h~48h, centrifuged, washed with anhydrous ethanol, and then vacuum dried at room temperature for 8-12h. The resulting yellow precipitate is NH2-MSN / Cur.

[0039] A fourth aspect of the present invention is to provide a curcumin-dual-response thermosensitive gel obtained by the preparation method described in the third aspect of the present invention.

[0040] A fifth aspect of the invention is to provide the use of curcumin-dual-responsive thermosensitive gel in the preparation of medicaments or health products for the prevention, relief and / or treatment of neurodegenerative diseases.

[0041] Furthermore, the neurodegenerative diseases mentioned are Alzheimer's disease and Parkinson's disease.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. This invention introduces PEG300 as a steric hindrance agent into the synthesis process of mesoporous silica, and combines it with high temperature and high rotation speed to reduce the nanodiameter of mesoporous silica, so as to achieve the synthesis of nanoparticles with a diameter of about 100nm under simple conditions, which are suitable for them to reach the intracranial cavity through the nasobrain pathway.

[0044] 2. After multiple screening and verification processes, this invention selectively modifies the surface of mesoporous silica nanospheres with TK and HA as targeting molecules to create a ROS-responsive targeted mesoporous silica nanocarrier, thereby improving the targeted distribution of drugs in Alzheimer's disease lesions and thus enhancing the therapeutic effect of the drugs. This invention employs MSN nanoparticles simultaneously modified with amino and azide groups. The amino groups are linked to a thioketal-mPEG-gated molecule via amide bonds, while the azide groups are linked to a hyaluronic acid targeting ligand via copper-free click chemistry. This avoids complex modification steps and interference between functional groups, ultimately resulting in MSN nanoparticles that target inflammation areas and are sensitive to ROS. While hyaluronic acid actively targets CD44 receptor-expressing cells (i.e., activated astrocytes and microglia) in neuroinflammatory processes, the modified thioketal cleaves in response to high levels of ROS under oxidative stress, enabling the modified MSN nanoparticles to actively target inflammatory lesions. Furthermore, when curcumin is loaded into TK and HA-modified MSN nanoparticles, this invention not only overcomes the problem of poor water solubility of curcumin, but also allows the modified MSN nanoparticles to actively target inflammatory lesions in Alzheimer's disease and responsively release curcumin within ROS-rich lesions. The synergistic effect of these two modifications promotes efficient accumulation of curcumin within Alzheimer's disease lesions, leading to rapid treatment of Alzheimer's disease.

[0045] 3. This invention incorporates inulin / poloxamer into dual-response MSN nanoparticles to form a semi-interpenetrating network structure, transforming the nanoparticles into a temperature-sensitive and adhesive thermosensitive gel formulation, thereby enhancing its mechanical strength. Under body temperature conditions, this prolongs the gel degradation time and drug retention time in the nasal cavity. Nasal administration allows the targeted nanoparticles to avoid being cleared by the nasal mucosa, thus providing sufficient time for them to directly enter the brain via the nasobrain pathway without crossing the blood-brain barrier. The addition of inulin to the poloxamer system in this invention has a dual function: it acts as a gelling raw material to stabilize the gel, forming a semi-interpenetrating network structure with the poloxamer system, significantly enhancing the mechanical strength and adhesion of the temperature-sensitive system; and it reduces the gelation concentration and phase transition time.

[0046] 4. The curcumin released from the curcumin-dual-response thermosensitive gel further prepared by this invention has strong pharmacological effects and is effective in preventing, regulating and treating Alzheimer's disease. It is fast-acting, has few toxic side effects, and is safe, showing good pharmaceutical prospects.

[0047] 5. The products used in this invention are safe to use, have a simple preparation process, and can also be made into a spray form. They are small in quantity, have good compliance, and are easy to use, thus making them easy to promote in clinical practice. Attached Figure Description

[0048] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration. These drawings, which constitute a part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0049] Figure 1 Fourier transform infrared (FTIR) spectra of N3 / NH2-MSN, HA-MSN, PEG-TK-MSN, and PEG-HA&TK-MSN.

[0050] Figure 2 This is a scanning electron microscope (SEM) image of PEG-HA&TK-MSN.

[0051] Figure 3 The diagram shows the color change reaction between azide-aminated mesoporous silica and curcumin ethanol solution.

[0052] Figure 4 This is an in vitro ROS-responsive drug release curve of curcumin-dual-response MSN.

[0053] Figure 5 This is a graph showing the temperature-sensitive changes in PHTMg.

[0054] Figure 6 This is a diagram showing the docking of HA with the CD44 receptor molecule.

[0055] Figure 7 The diagram shows the molecular dynamics simulation of HA and human CD44 receptor, where A: RMSD; B: RMSF; C: SASA; D: Rg; E: number of hydrogen bonds; F: average binding free energy.

[0056] Figure 8 For PEG-HA&TK-MSN / EB Gel in Aβ 1-42 Targeted concentration distribution of lesions in model mice.

[0057] Figure 9The pharmacodynamic results of each group of drugs on AD mice are as follows (phosphorylated Tau protein p-Tau, total β-amyloid Aβ, malondialdehyde (MDA), superoxide dismutase (SOD) activity, and interleukin-1β (IL-1β).

[0058] Figure 10 Animal behavioral experimental indicators include open field test indicators, where A: center time; B: total distance; C: Y-maze test, count of entering new arms; D: new object recognition test, recognition index (RI); E: tail suspension test, struggle time.

[0059] Figure 11 Visualization diagrams for animal behavioral experiments (Open Field experiment; Novel Object Recognition experiment; Y maze experiment).

[0060] Figure 12 Immunohistochemical images of hippocampal astrocyte activation in AD mice after intranasal administration (CA1 in horn 1; CA3 in horn 3; DG in dentate gyrus; GFAP immunohistochemistry, 100X).

[0061] Figure 13 The therapeutic effects of intranasal administration on the brain tissue of AD model mice (CA1 in horn ammonia 1 region; CA3 in horn ammonia 3 region; DG in dentate gyrus; Nissl staining, 100X).

[0062] Figure 14 The effects of PEG-HA&TK-MSN Gel on serum hematologic toxicity (ALT, AST, and BUN) in normal mice were investigated.

[0063] Figure 15 The toxic effects of PEG-HA&TK-MSN Gel administered intranasally on various organs and tissues of normal mice are shown in the diagrams (CA1 in the ammonia horn 1 region; CA3 in the ammonia horn 3 region; Cx in the cerebral cortex; DG in the dentate gyrus; nasal mucosa; liver; kidney; heart; spleen; lung; HE staining, 100X). Detailed Implementation

[0064] The preferred embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0065] The mass / volume concentration of the MSN solution in the specific embodiments of the present invention is illustrated by taking 1% (w / v) as an example. Other concentrations such as 0.5%-1.5% (w / v) are also applicable to the present invention.

[0066] Reagents used and their abbreviations: hexadecyltrimethylammonium bromide (Aladdin, CTAB, ≥99%), sodium hydroxide (NaOH), methanol, anhydrous ethanol, acetone, tetraethyl orthosilicate (Aladdin, TEOS, ≥98%), 3-aminopropyltriethoxysilane (Aladdin, APTES, ≥98%), 3-aminopropyltriethoxysilane (Aladdin, A... Z PTES (≥97%), hydrochloric acid, 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid (Aladdin, ≥95%), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (Aladdin, EDC·HCl, ≥98%), N-hydroxysuccinimide (Aladdin, NHS, ≥98%), dibenzocyclooctyne-amine (TCI, DBCO-NH2, ≥95%, CAS:1255942-06-3), poloxamer-407 (Aladdin, ≥98%) Aladdin (P407), Poloxamer-188 (Aladdin, P188), Inulin (In, Aladdin), ultrapure water used in the experiment was provided by the water purification system, MES buffer, PBS buffer, hyaluronic acid (Lon, HA, M=7kDa, ≥97%), methoxy polyethylene glycol (Aladdin, mPEG-NHS, M=750Da, ≥95%), dimethyl sulfoxide (Sinopharm), polyethylene glycol 300 (PEG300), curcumin (Cur, Maclean, 98%).

[0067] Example 1

[0068] The preparation methods for S1, MSNs, and N3 / NH2-MSN are as follows:

[0069] S1.1 MSNs: 1g CTAB and 480mL ultrapure water were added to a 500mL Erlenmeyer flask and stirred at 75℃ to dissolve. Then, 0.25mL PEG300 and 2mol / L NaOH (aq, 3.5mL) were added, and the mixture was stirred at 75℃ for 30min to form a template. Then, 5mL TEOS was slowly added, and the mixture was stirred for 2h. After centrifugation, washing with deionized water and ethanol three times each, the mixture was dried under vacuum at 50℃ for 12h to obtain a white precipitate. To obtain ordered mesoporous channels, the template was removed using an acidic methanol solution. 1g of the white precipitate was placed in 169mL of acidic methanol solution (160mL methanol, 9mL hydrochloric acid), sonicated for 15min to fully disperse it, and then stirred and heated at 60℃ for 12-14h to remove CTAB. After centrifugation, washing with deionized water and ethanol 3-5 times each, the mixture was dried under vacuum at 50℃ for 12h to obtain MSNs; mesoporous silica nanoparticles (MSNs).

[0070] S1.2 Azide-aminated mesoporous silica particles (N3 / NH2-MSN): 0.5 g MSNs were added to a mixture of 0.234 mL LAPTES and 0.126 mL A Z The white powder product obtained by stirring PTES in 50 mL of xylene under nitrogen at room temperature for 24 h, followed by centrifugation, washing three times with anhydrous ethanol, and vacuum drying at 50 °C for 12 h is N3 / NH2-MSN.

[0071] Preparation of S2 and TK-MSN:

[0072] S2.1 Add 18 mg of 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, 30.6 mg of EDC·HCl, and 18.4 mg of NHS to ice-cold MES buffer (1 mL, taken out at 3-8℃) at pH 4.5-5.0. Under nitrogen atmosphere and in the dark, stir at room temperature (20℃-25℃) to carry out the acylation reaction for 1 h to obtain the ROS-responsive crosslinking agent TKDA (i.e., the MES solution of TKDA).

[0073] S2.2 Dissolve 100 mg N3 / NH2-MSN in 50 mL of dimethyl sulfoxide solution. Add the TKDA obtained in the previous step dropwise (drop / 20 min, each drop is about 40~50 μl) to the N3 / NH2-MSN solution that has been ultrasonically dispersed (15 min). Stir the resulting solution at room temperature under nitrogen atmosphere in the dark for 24 hours. Collect the precipitate by centrifugation and purify it by washing it three times each with acetone and anhydrous ethanol. After vacuum drying the purified product at room temperature for 24 hours, the resulting white precipitate is TK-MSN.

[0074] Experimental tests showed that the TKDA infusion should be controlled at 1 drop per 15-25 minutes to facilitate the subsequent esterification of PEG and the other end of TK. This not only provides a ROS response but also effectively encapsulates the drug in the MSN, which is beneficial for drug release after gel formation.

[0075] Preparation of S3 and PEG-TK-MSN:

[0076] S3.1 Dissolve 100 mg TK-MSN in dimethyl sulfoxide (10 mL), add 19.2 mg EDC·HCl and 12 mg NHS to perform secondary activation on the remaining carboxyl groups on the TK chain, and react for 1.5 hours;

[0077] S3.2 Dissolve 100 mg mPEG-NH2 (2-5 kDa, preferably 2 kDa) in 50 mL of dimethyl sulfoxide. Add the reaction solution from step S3.1 dropwise to the mPEG-NH2 mixed solution and stir at room temperature under nitrogen cover in the dark for 24 hours (usually the reaction can be carried out for 24-36 hours). Collect the final product by centrifugation and wash it 3-5 times alternately with anhydrous ethanol and acetone to completely remove all unreacted reagents and byproducts. Freeze-dry at -50°C under vacuum for 24 hours to obtain the final product PEG-TK-MSN.

[0078] Preparation of S4, PEG-HA & TK-MSN:

[0079] S4.1 Dissolve 100 mg HA in 10 mL MES buffer (pH 5.5), add 57.5 mg EDC·HCl and 34.5 mg NHS, and stir at room temperature for 30 min to activate the carboxyl group of HA. Add 10 mg DBCO-NH2, adjust the pH of the solution to about 7.4 with NaOH, and stir at room temperature in the dark for 12 h. Transfer the final reaction solution into a dialysis bag (MWCO, 10 kDa), dialyze in ultrapure water for 24 h, pre-freeze the liquid in the dialysis bag at -80 °C, and then freeze-dry under vacuum at -50 °C for 24 h to obtain a white solid HA-DBCO.

[0080] S4.2 Add 100 mg of HA-DBCO obtained in S4.1 to a 100 mg PEG-TK-MSN suspension dispersed in 50 mL of dimethyl sulfoxide; stir and react at room temperature under nitrogen cover for 36 hours (usually 24-36 hours); collect the product by centrifugation, wash with ultrapure water and anhydrous ethanol alternately 3-5 times to remove unreacted HA-DBCO; freeze-dry at -50℃ for 24 hours to obtain PEG-HA&TK-MSN.

[0081] S5. Preparation of thermosensitive gel:

[0082] Place 1 mL of PEG-HA&TK-MSN solution (20 mg / mL, mixed in pre-cooled PBS buffer at 3°C) in an Erlenmeyer flask, then add poloxamer P407 (P407, 200 mg), poloxamer P188 (P188, 20 mg), and inulin (20 mg). Stir at room temperature until the poloxamer and inulin dissolve. Allow the mixture to stand at low temperature (3~8°C) for 24 hours (usually 18-24 hours), then freeze at -20°C for 12 hours and alternate between freezing and thawing at room temperature for at least 3 times. Finally, thaw at room temperature. The polymer chain segments will fully expand, forming a stable colloidal solution. This colloidal solution is the thermosensitive gel stock solution, namely the biresponsive MSN thermosensitive gel PEG-HA&TK-MSN gel (abbreviated as PHTMg).

[0083] Example 2

[0084] Preparation of S1, N3 / NH2-MSN / Cur:

[0085] Steps S1.1 and S1.2 are the same as in Example 1;

[0086] S1.3 Drug loading: 50 mg N3 / NH2-MSN was added to 50 mL of anhydrous ethanol solution of curcumin with a concentration of 10 mg / mL, and the pH was adjusted to 6.5 by adding MSE buffer solution. The mixture was stirred at room temperature in the dark for 24 h to allow it to be fully loaded. After centrifugation and washing three times with anhydrous ethanol, it was vacuum dried at room temperature for 8-12 h. The resulting yellow precipitate was N3 / NH2-MSN / Cur. Subsequent nano-surface modification was carried out according to the steps in Example 1.

[0087] The curcumin-ROS responsive nanoparticles (TK-MSN / Cur) were prepared by replacing N3 / NH2-MSN / Cur in step S2 with N3 / NH2-MSN / Cur, and the remaining steps were the same as in step S2.

[0088] Continue with step S3 to prepare PEG-TK-MSN / Cur;

[0089] Continue with step S4 to prepare curcumin-dual-responsive nanoparticles (PEG-HA&TK-MSN / Cur).

[0090] S5. Place 1 mL of curcumin-dual-responsive nanoparticle (PEG-HA&TK-MSN / Cur) solution (20 mg / mL, mixed in pre-cooled PBS buffer at 3°C) in an Erlenmeyer flask, then add poloxamer P407 (P407, 200 mg), poloxamer P188 (P188, 20 mg), and inulin (20 mg). Stir at room temperature until the poloxamer and inulin dissolve. Let the mixed solution stand at low temperature (10°C) for 24 hours (usually 18-24 hours), freeze at -20°C for 2 hours, and freeze and thaw at room temperature at least 3 times each. Thaw at room temperature. The polymer chain segments will fully expand to form a stable colloidal solution. This colloidal solution is the thermosensitive gel stock solution, namely curcumin-dual-responsive MSN-thermosensitive gel PEG-HA&TK-MSN / Cur gel (abbreviated as PHTcg).

[0091] like Figure 5 As shown, PEG-HA&TK-MSN / Cur Gel will transform into a gel state at a temperature of 34℃.

[0092] Example 3

[0093] 1. Fourier transform infrared spectroscopy analysis

[0094] Preparation of HA-MSN: MSNs were obtained in the same manner as in Example 1. 50 mg of MSNs were mixed with 0.126 mL of A. Z In a xylene solution of PTES, after stirring under nitrogen at room temperature for 12 h, the mixture was centrifuged, washed, and vacuum dried at room temperature for 6 h to obtain N3-MSN. 700 mg of HA was dissolved in 10 mL of MES buffer (pH 5.5), and 61.2 mg of EDC·HCl and 40 mg of NHS were added. The mixture was stirred at room temperature for 30 min to activate the carboxyl group of HA. 60 mg of DBCO-NH2 was added, and the pH of the solution was adjusted to approximately 7.4 with NaOH. The mixture was stirred at room temperature in the dark for 12 h. The final reaction solution was placed in a dialysis bag (10 kDa), dialyzed in ultrapure water for 24 h, and then the liquid in the dialysis bag was pre-frozen at -80 °C and then freeze-dried under vacuum at -50 °C for 24 h to obtain a white solid HA-DBCO. Add 100 mg of the obtained HA-DBCO to a suspension of 100 mg N3-MSN dispersed in 50 mL of dimethyl sulfoxide; stir and react at room temperature under nitrogen cover for 36 hours (usually 24-36 hours); collect the product by centrifugation, wash with acetone and anhydrous ethanol alternately 3-5 times to remove unreacted HA-DBCO; freeze-dry at -50 °C for 24 hours to obtain monomodified HA-MSN.

[0095] Preparation of PEG-TK-MSN: MSNs were obtained in the same manner as in Example 1. 50 mg of MSNs were mixed in 0.234 mL of APTES aqueous solution, stirred under nitrogen at room temperature for 12 h, centrifuged and washed, and then vacuum dried at room temperature for 6 h to obtain NH2-MSN. 36 mg of 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, 61.2 mg of EDC·HCl, and 40 mg of NHS were added to ice-cold MES buffer (1 mL, taken out at 3-8℃) at pH 4.5-5.0. Under nitrogen atmosphere and in the dark, the mixture was stirred at room temperature (20℃-25℃) for 1 h to prepare the ROS-responsive crosslinking agent TKDA. 100 mg of NH2-MSN was dissolved in 50 mL of dimethyl sulfoxide solution. The TKDA obtained in the previous step was added dropwise to the NH2-MSN solution that had been ultrasonically dispersed (15 min). The resulting solution was stirred at room temperature under nitrogen atmosphere and in the dark for 24 h. The precipitate was collected by centrifugation and purified by washing three times each with acetone and anhydrous ethanol. The purified product was dried under vacuum at room temperature for 24 h, and the resulting white precipitate was TK-MSN. 100 mg TK-MSN was dissolved in 2 mL of pre-cooled MES buffer (taken out at 3-8℃), and 61.2 mg EDC·HCl and 40 mg NHS were added to reactivate the remaining carboxyl groups on the TK chain for 1.5 hours. 100 mg mPEG-NH2 (2-5 kDa, preferably 2 kDa) was dissolved in 10 mL of borate buffer, and the pH of the solution was adjusted to about 8.0 with NaOH (the entire process was controlled at pH 8.0-8.5). The activated TK-MSN reaction solution was added dropwise to the buffer solution, and stirred at room temperature under nitrogen cover in the dark for 24 hours (usually the reaction can be carried out for 24-36 hours). The final product was collected by centrifugation and washed 3-5 times alternately with ultrapure water and anhydrous ethanol to completely remove all unreacted reagents and byproducts. The product was freeze-dried under vacuum for 24 hours to obtain the final product, monomodified PEG-TK-MSN.

[0096] N3 / NH2-MSN, HA-MSN, PEG-TK-MSN, and PEG-HA&TK-MSN were detected by infrared spectroscopy using the potassium bromide pellet method. Solid powder samples were mixed with potassium bromide powder in appropriate amounts, ground, pressed into thin pellets using a mold, and then tested. Results are as follows: Figure 1 As shown in the figure, analysis revealed that the present invention successfully synthesized the required PEG-HA&TK-MSN.

[0097] 2. Scanning electron microscopy analysis

[0098] PEG-HA&TK-MSN was examined under a scanning electron microscope. A 1cm long and 0.5cm wide conductive tape was attached to the sample stage. The protective film on the conductive tape was removed, and a small amount of powder was evenly spread onto the conductive tape. Any loose powder was blown off with a bulb syringe. The sample was then subjected to gold sputtering in a gold sputtering machine and observed. Results are as follows: Figure 2 As shown, the results indicate that the diameter of the synthesized nanocarriers is mainly distributed around 100 nm.

[0099] 3. Determination of drug loading rate of curcumin-dual-responsive MSN nanoparticles (PEG-HA&TK-MSN / Cur)

[0100] 1 mL of N3 / NH2-MSN solution was vacuum dried and the weight of the nanoparticle solid was recorded as 1 mg. Anhydrous ethanol solutions of curcumin with concentrations of 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, and 3 g / L were prepared. Then, 1 mL of curcumin solution was added to the nanoparticle solid for loading. PEG-HA&TK-MSN / Cur was then synthesized according to Example 2, and H2O2 solution was added, which was used to break the thioketal bonds. Finally, the mixture was sonicated three times, with each sonic extraction lasting 30 min. 1 mL of fresh ethanol solution was used each time. The three ethanol extraction solutions were transferred to a 10 mL volumetric flask and diluted to volume. The solution was filtered through a 0.45 µm microporous membrane to obtain the sample solution with the drug loading to be measured.

[0101] The curcumin content in the sample solution was determined by a UV-Vis spectrophotometer. Since the MSN component in the sample caused measurement interference at 254 nm, 430 nm was selected as the detection wavelength for curcumin, and the method is feasible under this wavelength condition.

[0102] The mass (m2) of curcumin in curcumin-dual-responsive nanoparticles was determined by UV-Vis spectrophotometry. The drug loading rate was calculated according to the following formula (1), and the encapsulation efficiency was calculated according to formula (2):

[0103] (1) Drug loading rate (%) = Mass of drug loaded in nanoparticles (m2) / Mass of drug-loaded nanoparticles (m1) × 100%

[0104] (2) Encapsulation efficiency (%) = Mass of drug loaded in nanoparticles (m2) / Total mass of drug-loaded nanoparticles (m3) × 100%.

[0105] Table 1 Results of drug loading and encapsulation efficiency determination

[0106]

[0107] Table 1 shows the experimental results: as the concentration of curcumin ethanol solution increases, its drug loading rate gradually increases and eventually stabilizes. The highest drug loading rate can reach 43%, indicating that the dual-response MSN of the present invention has a high drug loading capacity.

[0108] 4. Color change phenomenon of curcumin acid-base indicator

[0109] Curcumin's molecular structure contains phenolic hydroxyl groups and β-diketone structures, making it highly sensitive to environmental pH and a natural pH indicator. It appears bright yellow under acidic or neutral conditions. Under alkaline conditions, its complementary color changes to reddish-brown or dark brown. Aminated mesoporous silica provides this alkaline environment.

[0110] (1) Basicity of surface amino (-NH2) groups: Mesoporous silica surfaces are chemically modified to incorporate amino (-NH2) functional groups. These amino groups exhibit Brønsted base properties in anhydrous ethanol, accepting protons and increasing the local pH, thus exhibiting basicity. The reaction can be simplified as follows: .

[0111] (2) Interaction between curcumin and amino groups: When an ethanol solution of curcumin is added to aminated mesoporous silica, curcumin molecules rapidly enter the mesoporous channels through capillary action and come into close contact with a large number of amino functional groups on the inner wall of the channels. This places the curcumin molecules directly in a locally strongly alkaline environment, resulting in a visible reddish-brown color.

[0112] Discoloration phenomenon such as Figure 3 As shown, initially, curcumin is dissolved in ethanol in a neutral environment and appears bright yellow. When this solution is added to aminated mesoporous silica, curcumin molecules diffuse and adsorb into the mesoporous channels. The phenolic hydroxyl groups on the curcumin molecules interact with the amino groups on the silica surface, causing the phenolic hydroxyl groups to lose protons and transform into phenoxy anions. This change in molecular conjugation results in a red shift in the absorption spectrum, macroscopically manifested as a change in solution color from yellow to reddish-brown or dark brown.

[0113] 6. Determination of chemically accessible groups on the MSN surface

[0114] To quantify the number of modifiable groups on the surface of MSN, all groups on the MSN surface were converted to amino groups, i.e., NH2-MSN, and a ninhydrin test was performed. NH2-MSN (1 mg) was sonicated in ethanol (1 mL) at room temperature for 15 minutes. The resulting dispersion was mixed with ninhydrin chromogenic solution (20 mg / mL) in 2 mL of the reaction mixture and stirred at room temperature for 18 minutes. The absorbance at 570 nm was then analyzed, and the number of amines in NH2-MSN was determined using a standard curve. The result was 1.5 mmol / g, indicating that the number of modifiable groups on the MSN surface was 1.5 mmol / g.

[0115] 7. Study on in vitro ROS-responsive release of curcumin from curcumin-dual-response MSN-thermosensitive gel

[0116] (1) PEG-HA&TK-MSN / Cur was prepared according to the method in Example 2 and a release experiment was conducted.

[0117] (2) Phosphate-buffered saline (PBS) with pH=7.3 was selected as the release medium, and physiological environment simulation release was carried out under the condition of 34℃ in the microplate reader.

[0118] (3) Place 0.143 mg PEG-HA&TK-MSN / Cur in 400 μL of PBS buffer containing hydrogen peroxide (0 M, 50 mM, 100 mM, 200 mM) at a preheated temperature of 34 °C. Set the microplate reader to measure absorbance every 5 minutes from 0-1 h, every 0.5 h from 1 h-24 h, and every 1 h from 24 h-48 h. Shake for 10 s before each measurement, collect all absorbance, and determine the concentration of curcumin in the release solution according to the standard curve. The in vitro drug release performance is expressed as the cumulative drug release percentage. The cumulative drug release rate (%) is calculated according to the formula: Qn=(En×V / m cur )×100%.

[0119] Where: Qn is the cumulative drug release at the nth sampling; En is the drug concentration in the release medium at the nth measurement; V = 200 μL; n is an integer, n = 1, 2, 3, ...; m cur =0.043mg.

[0120] Drug release results as follows Figure 4 As shown, curcumin is released rapidly from 0 to 2 hours, but the release becomes slow and stable in the later stages, a release pattern that better meets the requirements for drug delivery into the brain. From 2 to 12 hours, the curcumin release curve is approximately linear, indicating a slow and stable release. Furthermore, as the H2O2 concentration in the buffer solution increases, the release rate of curcumin accelerates, the final height of the release curve increases, and the release rate increases, meeting the requirement for on-demand drug release to the lesion area.

[0121] Currently, the targeting issues in naso-brain drug delivery result in widespread drug release in the early stages, leading to a lack of lesion-specific targeting in the brain. The TK modification of this invention enables the drug to be released on demand and stably in the ROS microenvironment of the lesion. TK modification and encapsulation allow curcumin to be released in a ROS-responsive manner, which is more conducive to maintaining a stable drug concentration at the lesion site in the brain and avoiding adverse reactions caused by insufficient drug targeting.

[0122] 8. Test tube inversion method for dual-response thermosensitive gel (PHTMg)

[0123] Take a fixed mass of inulin and different dosage ratios of P407 and P188 (according to Table 2), and take different dosage ratios of P407 and P188 (according to Table 3). Add 1 mL of ultrapure water to each solution and stir to dissolve. Place the solutions in a refrigerator at 3-8°C for 12-24 hours, then freeze at -20°C for 12 hours and thaw at room temperature at least three times each. Thaw at room temperature. Place the resulting solutions in test tubes and measure the gel temperature of different formulations in a water bath at 25°C at a rate of 0.5°C / min. The dosage ratios selected for this invention are shown in Tables 2 and 3. Phase transition temperatures were measured according to these ratios.

[0124] Table 2. Thermosensitive Gel Tube Inversion Study: Dosage Ratio of In / P407 / P188, Phase Transition Temperature, and Phase Transition Time

[0125]

[0126] Table 3. Dosage ratio and phase transition temperature of P407 / P188 in the test tube inversion study.

[0127]

[0128] Keeping the P407 content in the gel system constant, the gelation phase transition temperature continuously increased with increasing P188 content; conversely, keeping the P188 content constant, the gelation phase transition temperature continuously decreased with increasing P407 content. Compared to pure poloxamer hydrogel, the required gelation concentration after adding inulin was lower. As a hydrophilic polysaccharide, inulin attracts water molecules surrounding the polyoxyethylene chains of P407. This may be related to the dehydration effect and size exclusion of inulin, making micelles easier to form and compact, thus triggering gelation at lower concentrations. By adjusting different ratios of P407 and P188, a thermosensitive gel formulation meeting the requirements of nasal administration was obtained. The optimal ideal ratio of inulin, P407, and P188 was 2:20:2.

[0129] 9. Effects of different concentrations of inulin on the degradation time and gelation time of In / P407 / P188 composite hydrogels

[0130] Add different concentrations of inulin (prepared according to Table 4), 200 mg of P407, and 20 mg of P188. Place test tubes containing 1 mL of the mixed solution in a 33°C constant temperature water bath and observe the changes in the mixture. When the solution stops flowing after the vial is inverted, it is considered that the solution has formed a hydrogel, and the shortest time required is the gelation time.

[0131] In the in vitro degradation experiment, hydrogel blocks of the same volume were immersed in 30 mL of PBS, kept at 37 °C, and shaken at 100 r / min. The state of the hydrogel in PBS was observed until it was completely degraded, and the time was recorded.

[0132] Table 4. In vitro degradation and gelation experiments

[0133]

[0134] As shown in Table 4, the degradation time and gelation time increased with the increase of inulin concentration. This may be related to the semi-interpenetrating network structure formed by inulin and poloxamer system and the "salting out" effect of inulin, which compensates for the weak mechanical strength of pure poloxamer hydrogel, thus prolonging the degradation time of hydrogel and extending its presence in the nasal cavity.

[0135] 10. Hydrogel Tensile Adhesion Test

[0136] To evaluate the adhesion ability of hydrogels, we used a motorized vertical dual-column test bench to test the adhesion strength of hydrogels with different compositions to rubber in the vertical direction. The specific steps are as follows: The corresponding hydrogels (prepared according to Table 5) were prepared and solidified at 37°C. The hydrogels were then adhered between the end faces of two parallel rubber sheets. The other ends of the two rubber sheets were fixed in upper and lower clamps, respectively. The equipment was started, and vertical stretching was applied until the adhesive interface failed. The maximum tensile force at failure was recorded.

[0137] Table 5 Maximum adhesion force of different samples

[0138]

[0139] As shown in Table 5, compared with pure poloxamer thermosensitive hydrogel, the addition of inulin increases the adhesion of poloxamer thermosensitive hydrogel, which can significantly enhance the adhesion of the gel, allowing it to adhere to objects for a long time and thus not be cleared by the nasal cavity, achieving long-term drug release. However, the adhesion is weaker than that of 50% pure inulin hydrogel.

[0140] Example 4: Molecular docking results of sodium hyaluronate and CD44 receptor

[0141] The CD44 protein sequence was searched on the Uniprotain website. The found sequence (P16070) was imported into the SWISS-MODLE website to find the optimal structure and saved as a PDB file. Sodium hyaluronate was searched in a small molecule database and downloaded in SDF format. The ligand was optimized using DS software, and the optimized structure was saved as a PDB file for later use. The CD44 protein file was imported into PYMOL software, redundant ligands and water molecules were removed, and the file was saved as a PDB file for later use. The protein was hydrogenated and charged, and set as a receptor, and saved as a PDBQT file for later use. Small molecules were hydrogenated and charged, and set as ligands. Twist angles and twist bonds were checked, and the files were saved as PDBQT files for later use. Since the binding pocket region was unknown, the grid was set to the entire protein region for ligand binding, and this operation was performed. After the grid setting was completed, 50 molecular Autodock dockings were performed. After docking is completed, the conformation with the highest repeatability and best binding energy is selected as the output conformation, and PYMOL is used to visualize it and export the 3D structure and surface model.

[0142] Molecular docking results showed that the binding energy of HA with mouse CD44 was -6.2 kJ / mol (< -6 kJ / mol); and the binding energy with human CD44 was -7.2 kJ / mol (< -7 kJ / mol). Figure 6 As shown in the figure, all samples exhibit good binding activity, indicating a good docking effect. HA is highly feasible as a target molecule for the CD44 receptor.

[0143] Example 5: Molecular dynamics results of sodium hyaluronate and human CD44 receptor

[0144] Gromacs 2025 was used to perform a 100 ns molecular dynamics simulation of the protein-ligand complex. The specific procedure was as follows: the protein CD44 was constructed using AMBER14SB force field parameters, and the topology of the sodium hyaluronate ligand was constructed using GAFF2 force field parameters. The protein and ligand topology files were merged to ensure no atomic type conflicts. Periodic boundary conditions (PBC) were used, placing the complex in a cubic box with a minimum distance of 1.2 nm from the complex surface to the box wall. The TIP3P water model was used as the solvent, and Na⁺ / Cl⁻ counterions were added to neutralize the net charge of the system. Energy minimization was performed first, followed by 1,000,000 steps of isothermal and isochoric ensemble equilibrium and isothermal and isobaric ensemble equilibrium, with a coupling constant of 0.1 ps and a simulation duration of 2 ns. The temperature / pressure coupling parameters were the same as in the equilibrium phase, and the trajectory was saved every 1,000 steps for subsequent analysis. Finally, a free molecular dynamics simulation was run, consisting of 50,000,000 steps at a step size of 2 fs, for a total duration of 100 ns. Finally, molecular dynamics simulations were performed using Gromacs 2025 at a constant temperature (310 K) and constant pressure (1 bar) for a total duration of 100 ns. The binding free energy after system stabilization was calculated using MMPBSA. The following parameters were calculated using GROMACS: root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), number of protein-ligand hydrogen bonds, and mPBSA (mean binding free energy, residue degradation energy).

[0145] Test results are as follows Figure 7 As shown: RMSD: After 10 ns, the RMSD value tends to stabilize, indicating that the protein has reached a stable state after ligand binding. The ligand does not move significantly within the binding site, indicating relatively stable binding. RMSF: Most residues have low RMSF values, indicating a relatively stable protein structure. The N-terminus has a higher RMSF, possibly indicating a flexible region of the protein. SASA: After 10 ns, SASA decreases, indicating that the protein more tightly encapsulates the ligand, further proving stable binding. Rg: After 10 ns, the Rg value decreases and tends to stabilize, indicating that the protein structure becomes more compact after ligand binding. Number of hydrogen bonds: After 10 ns, the number of hydrogen bonds stabilizes, indicating that the protein and ligand have formed stable hydrogen bond interactions. Average binding free energy: The total binding free energy is -21.62 kcal / mol, indicating that the ligand and protein can bind spontaneously, and the bound protein is thermodynamically stable. The energy values ​​of residues such as VAL-148, ARG-150, and GLY-152 are relatively small negative values, indicating that these residues contribute relatively strongly to the stability of protein-ligand interactions, ultimately demonstrating the good stability of HA docking with the CD44 receptor.

[0146] Example 6: Disease Targeting Validation Experiment of PEG-HA & TK-MSN / EB Gel

[0147] Because curcumin itself is metabolized, Evans blue (EB), a multifunctional azo blue dye widely used in medical and biological research, rapidly binds tightly to surrounding albumin upon release, forming a large molecular complex that is "anchored" at the release site, resisting diffusion, and has a maximum absorption wavelength of 610 nm, allowing for quantitative measurement. Therefore, replacing curcumin with Evans blue to load dual-response nanoparticles allows for UV observation of the content of Evans blue-labeled nanoparticles in the left and right hemispheres of the brain, and analysis of whether TK and HA modifications can increase the lesion targeting of the nanoparticles.

[0148] 1. Preparation of Evans Blue Nanoparticles (PEG-HA & TK-MSN / EB):

[0149] S1: Prepare azide-aminated mesoporous silica using the same method as in Example 1.

[0150] S2: Weigh 0.1g of N3 / NH2-MSN and add it to 10mL of anhydrous ethanol to prepare a 1% MSN solution. Stir magnetically at 4℃ overnight before use.

[0151] S3: Except for weighing 0.5 mg of Evans blue, which replaces curcumin with Evans blue, the rest of the steps are the same as in Example 2 to prepare PEG-HA&TK-MSN / EB.

[0152] 2. Preparation of Aβ model mice

[0153] 2A, Aβ 1-42 Solution: Aβ 1-42 (Qiangyao Biotechnology, >95%) Dissolve in sterile physiological saline to a final concentration of 410 pmol / 5u1, aliquot and freeze for later use. Before use, incubate at 36℃ for 1 hour to allow it to reach the desired aggregate state.

[0154] 2B. Select healthy male mice, around 4 months old, weighing 30-35g.

[0155] 2C. Mouse anesthesia: Before surgery, anesthetize the mouse by inhaling oxygen containing isoflurane (flow rate 1L / min, containing 5% (v / v) isoflurane) until the corneal and toe reflexes disappear. Alternatively, anesthesia can be achieved by intraperitoneal injection of afodin.

[0156] 2D. Mouse Treatment: Insert fixation rods into both external auditory canals to fix the mouse's head on a stereotaxic apparatus in a prone position. Shave the hair from the top of the mouse's head, disinfect with iodine, and then make an incision with a scalpel. Select the CA1 region of the hippocampus on one side as the directional injection area, located 2.0 mm posterior to the anterior fontanelle and 2.0 mm lateral to the left midline; that is, only the CA1 region of the left hippocampus is injected to create a lesion on one side of the brain, with no lesion on the right side, serving as a control. Mark the injection point, drill a hole at the marked location using a dental drill, slowly penetrating the skull until a feeling of emptiness is felt. Insert a micropipette vertically into the dura mater to a depth of 2.0 mm, and slowly inject the prepared Aβ1-42 solution at a volume of 2.5 μL (the control group received the same volume of sterile saline). The injection rate is 0.5 μL / min. After injection, leave the needle in place for 2 minutes to ensure sufficient diffusion of the solution into the local hippocampal tissue, and then slowly withdraw the needle. The bone wound was sealed with bone wax, the incision was sutured, and then 0.2 mL of gentamicin injection was administered to the suture site for anti-inflammatory purposes, thus completing the modeling process. Approximately 20 minutes after the procedure, the mice awoke and were observed to see if they could forage and drink normally.

[0157] One day after successful model establishment, mice were administered the drug intranasally, with 10 μL administered to each nostril. Two hours after administration, the mice were sacrificed, their hearts were perfused with PBS, and brain tissue was harvested and divided into left and right hemispheres. The left and right hemispheres were then homogenized in 1 mL of 50% TCA. The absorbance of both hemispheres at 610 nm was measured using a UV-Vis spectrophotometer.

[0158] result Figure 8 The results showed that Evans blue had a much higher absorbance in the left brain than in the right brain, indicating that the content in the left brain was much higher than that in the right brain. This suggests that loading PHTMg with the drug can increase the distribution of the drug in the lesions of AD mice, thereby enhancing the anti-inflammatory, antioxidant and neuroprotective effects of the drug in the lesions.

[0159] Example 7: Efficacy test of curcumin-dual-response MSN-thermosensitive gel in Alzheimer's disease.

[0160] 1. Establishment of AD model mice

[0161] A combined D-galactose and aluminum trichloride modeling method was used. D-galactose and AlCl3 solutions were prepared using physiological saline. Mice were intraperitoneally injected with D-galactose (200 mg / kg) once daily, while simultaneously receiving AlCl3 solution by gavage (40 mg / kg), for 4 consecutive weeks. This completed the modeling process.

[0162] 2 Experimental Groups

[0163] The experiment was divided into 6 groups, with 5 mice in the PHTMg group and 10 mice in each of the other groups, for a total of 55 mice: The specific grouping is as follows: (1) Blank control group (N): C57BL / 6 mice, homogenized and given an equal amount of physiological saline; (2) Model group (M): AD mice, homogenized and given an equal amount of physiological saline; (3) HA nanoparticle thermosensitive gel (Hcg): AD mice, homogenized and given Hcg via nasal cavity; (4) TK nanoparticle thermosensitive gel (PTcg): AD mice, homogenized and given PTcg via nasal cavity; (5) HA and TK nanoparticle thermosensitive gel (PHTcg): AD mice, homogenized and given PHTcg via nasal cavity; (6) PHTM unloaded particle thermosensitive gel (PHTMg): AD mice, homogenized and given PHTMg via nasal cavity.

[0164] The effective concentration of curcumin in nasal administration was 5 mg / kg. In subsequent experiments, the amount of curcumin in the drug-loaded nanoparticles was maintained at 5 mg / kg in all groups. Curcumin was administered bilaterally at intervals, 5 µL per nostril, for 4 consecutive weeks. The model group and the control group received saline nasal administration.

[0165] 3 Experimental Procedure

[0166] The Y-maze test was performed on day 14 of drug treatment, the open field test on day 20, the novel object recognition test on day 25, and the tail suspension test on day 28 to preliminarily evaluate the efficacy of drug treatment through behavioral experiments. On day 29, mice in each group were anesthetized with 1.25% tribromoethanol (125mg / 100g). Blood was collected from the N group and the PHTMg group, and tissues from the brain, nasal mucosa, heart, liver, spleen, lung, and kidney were collected for biochemical indicators and pathological section analysis. Other groups were perfused systemically through the heart with PBS buffer (pH 7.4). When the buffer flowing from the heart was almost clear, five mice from each group were randomly selected and fixed with 4% paraformaldehyde until the mice's tails were raised. The mice were then removed and placed in a mouse-specific brain fossa. Coronal sections of approximately 7 slices were prepared at a thickness of 2mm from the anterior and posterior to the visual crossover point. Carefully remove brain slices from the cerebral fossa, immerse them in formalin solution, and store them protected from light for subsequent Nissl staining and immunohistochemistry of paraffin sections. The remaining five samples were removed using the same procedure for subsequent ELISA and oxidative stress marker detection.

[0167] Example 8: Animal behavioral experiments on the preliminary therapeutic effects of each group

[0168] (1) Y-maze: On day 14 of treatment, the Y-maze consisted of three arms with lengths of 34 cm, 10 cm, and heights of 10 cm, namely the starting arm, the new arm, and the other arms. One hour before the experiment, the experimental animals were placed in the testing room to acclimatize to the environment. At the start of the test, the new arm was blocked by a barrier, and the mice were placed in from the starting arm. After 5 minutes of free exploration in the starting arm and the other arms, the mouse movement trajectory was recorded. After 30 minutes, the mice were placed in the starting arm, the new arm was opened, and the mice were explored for 5 minutes, and the mouse movement trajectory was recorded. The number of times the mice explored the new arm was counted.

[0169] (2) Novel Object Recognition: After 20 days of treatment, we assessed cognitive abilities using the NOR test. On days 18 and 19 post-surgery, mice were allowed free exploration of an empty box (40 × 40 × 40 cm) for 5 min / day for 2 days as an adaptation period. On day 20, mice were allowed to explore two identical cubic objects located at the left and right corners of the black box. The exploration phase lasted 5 minutes. The correct cubic object was then replaced with a new spherical object. During this phase, a 5-minute performance was conducted using a video analysis system. Cognitive abilities were analyzed using the Recognition Index (RI), where RI = Novel object exploration time / (Novel object exploration time + Familiar object exploration time).

[0170] (3) Open Field Test: The open field test utilizes rodents' natural aversion to open spaces to assess general motor activity and anxiety in rodents. It was conducted on day 25 of treatment in a closed grid arena (40 cm × 40 cm × 40 cm) under dim lighting. The floor of the box was divided into nine squares, each approximately 13.3 cm × 13.3 cm in size, with the center square designated as the central area. Each mouse was gently placed on the central square and allowed to explore the arena spontaneously without interference. This spontaneous activity was recorded for 10 minutes. The total exploration time in the central area indicated anxiety / depression, and the total distance traveled was a measure of motor activity. The total distance traveled and the center time were recorded.

[0171] (4) Tail Suspension Test: On day 28 of drug administration, we used the tail suspension test to assess the degree of depression in mice. In short, mice were moved to a quiet testing room at least one hour before the test to acclimatize, maintaining low light and a room temperature of 20-25°C. The equipment was thoroughly cleaned with 75% ethanol or similar solution before and after the experiment to eliminate odors. The mice were secured in the tail suspension box with medical tape 1-2 cm from the tail tip, ensuring the tape was secure and did not injure the skin. The mice were suspended headfirst, with their noses approximately 30 cm from the ground, ensuring they could not touch any support. The camera was started and recorded continuously for 6 minutes. We recorded the time the mice struggled.

[0172] The results are as follows Figure 10 and Figure 11 As shown, the Y-maze test results indicated that, compared with the control group, the number of times the model group mice entered the new arm was significantly reduced, suggesting that the spatial working memory of the model group animals was impaired. After treatment with Hcg, PTcg, and PHTcg, the number of times they entered the new arm increased, with the Hcg and PHTcg groups showing statistically significant differences compared with the model group. In the new object recognition test, the control group mice showed a clear exploratory preference for new objects; the recognition index of the model group mice was significantly reduced, with a statistically significant difference compared with the control group (P < 0.001). After intervention with Hcg, PTcg, and PHTcg, the recognition index recovered somewhat, with the PHTcg group showing a significantly higher index than the model group. The open field test results showed that the total movement distance of the model group mice was significantly shorter than that of the control group, and the dwell time in the central area was significantly reduced. These results indicate that the model group animals exhibited significant anxiety-like behavior and decreased spontaneous activity ability. Compared with the model group, the Hcg, PTcg, and PHTcg groups showed increased total movement distance and increased dwell time in the central area, with the PHTcg group showing statistically significant differences. In the tail suspension test, the cumulative immobility time of mice in the model group was significantly longer than that in the control group (P < 0.001), suggesting that the animals in the model group exhibited significant depressive-like behavior. The immobility time of the drugs Hcg, PTcg, and PHTcg was statistically significant compared to the model group. (e.g.) Figure 10 As shown, each group was compared with group M (where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001), indicating that Hcg, PTcg, and PHTcg can enhance the learning and memory abilities of mice and have a neuroprotective effect, and PHTcg is more effective than the modified particles alone.

[0173] In summary, the results of the Y-maze, open field test, novel object recognition test, and tail suspension test all indicate that PHTcg, compared with gels prepared by modifying nanoparticles with HA or TK alone, is more effective in improving impaired spatial memory and learning abilities, and alleviating anxiety and depressive behaviors.

[0174] Example 9 Nissl staining of neuronal damage in each group

[0175] Nissl bodies are an important site of protein synthesis in neurons. When neurons are stimulated, the number of Nissl bodies within the cell is significantly reduced. Nissl staining results are as follows: Figure 13As shown, in the control group, the hippocampus cells in the CA1, CA3, and DG regions exhibited regular cell morphology, neat arrangement, tight layering, and clearly visible nuclear membranes. Nissl bodies were large and abundant, appearing as dark blue granules or tiger stripes. Conversely, in the M group, cell morphology was disordered, the nuclear membrane was unclear and irregular, Nissl bodies were blurred, and their number was reduced or even absent. After intervention with Hcg, PTcg, and PHTcg, Nissl bodies showed varying degrees of reversal and improvement in both shape and number, indicating that Hcg, PTcg, and PHTcg promote neuronal regeneration and have varying degrees of protective effects on neurons, thereby enhancing the learning and memory abilities of mice and ultimately alleviating AD symptoms. Among these, the PHTcg Nissl bodies were similar in shape and number to the control group, indicating that PHTcg promotes neuronal regeneration more effectively and has a stronger protective effect on neurons than Hcg and PTcg.

[0176] Example 10: GFAP immunohistochemistry of neuroinflammatory activation in each group

[0177] Tissue fixation: Immediately after sampling, immerse the brain tissue in a sufficient volume (20 times its volume) of 4% paraformaldehyde. Fixation time is typically 36 hours to preserve antigenicity and tissue structure. Dehydrate using a gradient of alcohols (70%, 80%, 95%, 100%), clear with xylene, and finally impregnate in molten paraffin to ensure complete paraffin penetration. Place the paraffin-impregnated tissue blocks in an embedding cassette to cool and solidify. Continuously section using a microtome, typically to a thickness of 3-5 micrometers. Flatten the sections in warm water (37°C), then lift them with a glass slide and bake in a 60°C oven for 20 minutes to ensure tight adhesion. Immerse in xylene I and II for 10-15 minutes each to completely remove paraffin. Rehydrate using a gradient of alcohols: 100% → 95% → 80% → 70% alcohol, immersing for 3-5 minutes each, and finally soaking in PBS buffer. Immerse the sections in pH 6.0 citrate buffer and heat in a microwave for 15 minutes. After heating, allow to cool naturally to room temperature. Incubate with 3% hydrogen peroxide (H2O2) solution at room temperature for 10-15 minutes, then rinse with PBS. Finally, block with 5% BSA solution at room temperature for 20-30 minutes. Discard the blocking solution and directly add the GFAP primary antibody working solution prepared with antibody dilution buffer, completely covering the tissue. Incubate overnight at 4°C. Place the slide horizontally in a humidified chamber overnight. After primary antibody incubation, wash three times with TBS buffer, 5 minutes each time. Add HRP-labeled anti-goat IgG. Incubate at room temperature for 60 minutes. Dilute the secondary antibody according to the instructions. Wash three times with TBS buffer, 5 minutes each time, to thoroughly remove unbound secondary antibody. Add DAB (diaminobenzidine) chromogenic solution to the tissue according to the instructions, and observe under a microscope in real time. When the specific brownish-yellow signal is clear and the background is unstained, immediately immerse the slide in tap water to stop the reaction. Stain the cell nuclei with hematoxylin for about 1 minute, then rinse with running water to return to blue. This makes the cell nuclei blue, forming a sharp contrast with the positive signal. The sections were dehydrated sequentially with a gradient of alcohols (70%, 80%, 90%, 95%, 100%). Clear the sections with xylene for 5 minutes each (steps I and II). After removing the sections from the xylene, a drop of neutral resin was placed on each tissue area, covered with a coverslip, and allowed to air dry. The sections were then observed under a light microscope. Positive signals were located in activated astrocytes; the proliferation and bioactivity of activated astrocytes in each group were compared.

[0178] Glial fibrillary acidic protein (GFAP) is a marker of reactive astrocytes, and astrocyte activation is a key and early event in the pathological process of Alzheimer's disease (AD). The expression level of GFAP can directly reflect the intensity of astrocyte-mediated neuroinflammatory response in the brain. Figure 12Immunohistochemical results showed that the astrocytes in the blank group were in a resting state, characterized by small cell bodies, thin processes, and abundant branching; while the astrocytes in the model group were in a reactive activated state, characterized by significantly enlarged cell bodies, thickened and elongated processes, deeper staining, and increased number, exhibiting typical pathological features of "astrocytosis." After intervention with HCG, PTcg, and PHTcg, the number of GFAP-positive cells in CA1, CA3, and DG regions decreased to varying degrees, and cell hypertrophy and thickened processes improved. The staining characteristics of the PHTcg group were similar to those of the N group, indicating that PHTcg was more effective than HCG and PTcg in alleviating the aggravation of neuroinflammation and exerting an anti-inflammatory effect.

[0179] Example 11 Effects of intranasally administered thermosensitive hydrogel on biochemical parameters in AD mice

[0180] Follow the instructions for the ELISA kits, including the p-Tau ELISA kit (YP-mAb-17775), Aβ ELISA kit (INS-2011608), and IL-1β ELISA kit (ZY Biotech, ZY-E6309M), to calculate the levels of p-Tau, Aβ, and IL-1β in brain tissue.

[0181] AD is a complex neurodegenerative disease. Abnormal accumulation of Aβ, hyperphosphorylation of Tau protein, and the generation of neuroinflammation ultimately induce apoptosis of nerve cells. Figure 9The ELISA results showed that in the AD mouse model, the levels of Aβ, p-tau, and IL-1β in the hippocampus were significantly increased. After treatment with Hcg, PTcg, and PHTcg, the Aβ levels in each group were (37.73±4.69), (35.76±3.26), and (18.07±2.32); the p-tau levels were (376.7±82.89), (369.7±94.81), (179.1±41.15), and (154.9±10.49); and the IL-1β levels were (161.7±34.44), (107.2±49.34), and (46.71±11.01). Reversing the levels of Aβ, p-tau, and IL-1β in brain tissue indicated that Hcg, PTcg, and PHTcg could reduce Aβ deposition and Tau protein hyperphosphorylation in AD mice, alleviating neuronal apoptosis and neuroinflammation. Among these, the PHTcg group showed the closest correlation with the N group after treatment, indicating its optimal efficacy in treating AD mice. (Comparisons between each group and the M group, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; comparisons between the Hcg and PTcg groups and the PHTcg ... # p < 0.05 ## This indicates that p < 0.01. ### (Indicates p < 0.001)

[0182] Example 12 Effect of intranasally administered thermosensitive hydrogel on oxidative stress markers in AD mice

[0183] The MDA (malondialdehyde) content detection kit (Dakewei, catalog number 8013071) and the superoxide dismutase (SOD) activity detection kit (Box Shenggong, catalog number AKAO001C-50S) were used to calculate the MDA content and SOD activity in brain tissue.

[0184] AD is a complex neurodegenerative disease in which oxidative stress plays an important role in the pathological process, ultimately inducing apoptosis of nerve cells. Figure 9The results of the oxidative stress experiment showed that in the AD mouse model, the MDA content in brain tissue was significantly increased and the activity of superoxide dismutase (SOD) was significantly decreased. After treatment with Hcg, PTcg, and PHTcg, the MDA values ​​of each group were Hcg (61.61±22.98), PTcg (35.29±13.33), and PHTcg (19.93±3.18); the SOD activities of each group were Hcg (14.43±1.699), PTcg (10.93±1.148), and PHTcg (19.93±3.18). g (29.43±7.875), all three significantly reversed MDA content and superoxide dismutase (SOD) activity. Notably, the PHTcg group had lower MDA content than the N group (30.11±2.31) and even higher SOD activity, which may be related to the increased local concentration of curcumin in the lesions, activating the endogenous antioxidant system and exerting its antioxidant effect. This indicates that PHTcg, compared to Hcg and PTcg, is more effective in reducing MDA content and increasing SOD activity in AD mice, thus alleviating the exacerbation of oxidative stress. (Comparison between each group and the M group, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001; comparison between Hcg and PTcg groups and the PHTcg ... # p < 0.05 ## This indicates that p < 0.01. ### This indicates that p < 0.001.

[0185] Example 13 Evaluation of the irritation of the nasal mucosa and the safety of various organs of the nasal delivery system of a dual-response thermosensitive hydrogel in AD mice.

[0186] The following method was used to stain the brain, nasal mucosa, heart, liver, spleen, lung, and kidney tissues of the N group and the PHTMg group with hematoxylin and eosin (H&E): the tissue fixed sections were dewaxed according to the specific steps in Example 10, and stained with the HE kit.

[0187] To investigate the effects of PHTMg on the histological morphology and neuronal damage of the hippocampus and cortex, the results are as follows: Figure 15As shown, in the control group, the pyramidal cell layer in the CA1 region of the hippocampus showed 2-3 layers of tightly packed pyramidal cells with clear outlines, large and round nuclei with fine chromatin, and prominent nucleoli; no neuronal condensation was observed. In the CA3 region of the hippocampus, pyramidal cells were larger than those in CA1, arranged slightly more loosely but with clear layers, and obvious apical dendrites extending into the radial layers. In the dentate gyrus (DG), the granular cell layer was densely packed in a "V" shape, with small and densely packed cells, little cytoplasm, and round, deeply stained nuclei. The cortex was clearly layered, and neurons were arranged in a columnar pattern and evenly distributed. After intervention with unloaded PHTMg, the number and morphology of neurons in AD mice were similar to those in the control group, indicating that the dual-response thermosensitive hydrogel drug delivery system had no significant toxic side effects on the central nervous system within 4 weeks.

[0188] To investigate whether PHTMg irritates the nasal mucosa within the nasal cavity, sections of nasal mucosal epithelial ciliated cells from the N group and the PHTMg group are shown below. Figure 15 As shown in the figure, the cell nucleus structure is clear and intact, the ciliated cells are densely distributed and neatly arranged, and there are no obvious bleeding points or damage to the nasal mucosal cells. The results indicate that the nasal delivery of the dual-response thermosensitive hydrogel drug delivery system does not irritate the nasal mucosal epithelial ciliated cells.

[0189] To investigate whether PHTMg causes toxicity and damage to tissues and organs in vivo, the microstructures of the heart, liver, spleen, lungs, and kidneys were observed. Figure 15 As shown in the image, the myocardial fibers are neatly arranged with clearly visible striations; the myocardial cells have uniform cytoplasm and oval nuclei located centrally; there is no congestion, edema, or inflammatory cell infiltration in the interstitium. The hepatic cords of the liver are arranged radially around the central vein, and no dilation or stenosis is observed in the hepatic sinusoids; the hepatocytes have abundant, eosinophilic cytoplasm, round nuclei, and clearly defined nucleoli; no hydropic degeneration, fat vacuoles, or necrotic foci are observed. The white pulp (splenic nodules) and red pulp (splenic cords and sinusoids) of the spleen are clearly demarcated, and the germinal centers of the splenic corpuscles are normally active; no obvious congestion or abnormal cell accumulation is observed in the red pulp and splenic sinusoids. The alveolar walls are thin and intact, and the alveolar cavities are clean, without exudate or inflammatory cells; the pseudostratified ciliated columnar cells of the bronchiolar epithelium are neatly arranged, and there is no detached material or mucus plug in the lumen. The glomeruli were uniform in size and had a normal cell count; no narrowing or dilation was observed in the Bowman's capsule. The brush border of the proximal tubules was intact, and the lumen of the distal tubules was patent, with no protein casts or crystals observed within the lumen. The results indicate that PHTMg did not cause damage to these tissues, suggesting that this dual-response thermosensitive gel drug delivery system has a certain degree of safety within the given dosage range and time period.

[0190] To investigate whether PHTMg causes toxicity and damage to tissues and organs in vivo, we used biochemical assays to detect ALT, AST, and BUN levels in plasma samples from each group. Blood samples were centrifuged at 3000 rpm for 10 minutes, and the plasma was analyzed according to the standard procedures of the reagent kit manufacturers. The kits used were: ALT (Nanjing Jiancheng, catalog number C009-2-1), AST (Nanjing Jiancheng, catalog number C010-2-1), and BUN (Nanjing Jiancheng, catalog number C013-3-1). Figure 14 As shown in the figure, the plasma concentrations of ALT, AST, and BUN in group N and group PHTMg are similar and have no significant statistical significance, indicating that no damage to cardiac, hepatic, or renal function was caused. This suggests that the dual-response thermosensitive hydrogel drug delivery system has a certain degree of safety within the given dosage range and time.

[0191] In summary, the thermosensitive hydrogel described in this application, constructed with inulin, poloxamer 407 (P407), and 188 (P188) and a dual-response MSN, yielded a nasally administered PHTMg without significant toxic side effects within the specified dosage range and time. Animal behavioral experiments showed that continuous intranasal administration of PHTcg significantly improved AD in mice. Nissl staining results showed that Hcg, PTcg, and PHTcg all significantly improved the number and morphology of neurons in the CA1 and CA3 regions of the hippocampus and the cerebral cortex of mice, with PHTcg showing similarities to the control group, indicating that PHTcg has a more protective effect on neurons than HA or TK modification alone. ELISA results showed that after 4 weeks of continuous treatment with Hcg, PTcg, and PHTcg, the levels of Aβ and p-Tau in brain tissue and neuroinflammation were significantly reduced, with PHTcg levels being closest to the control group, indicating that PHTcg... Compared to HA or TK modifications alone, Tcg alleviated AD symptoms and showed better therapeutic effects for AD. Immunohistochemical results showed that Hcg, PTcg, and PHTcg significantly reduced the number and morphology of activated inflammatory cells (astrocytes and microglia) in the CA1, CA3, and DG regions of the mouse hippocampus, exhibiting neuroanti-inflammatory effects. The PHTcg group was similar to the control group, indicating that PHTcg has a more protective effect on neurons than HA or TK modifications alone. Oxidative stress indicators showed that Hcg, PTcg, and PHTcg significantly improved the degree of oxidative stress in mouse intracranial brain tissue, demonstrating antioxidant effects. This indicates that PHTcg has a more protective effect on neurons than HA or TK modifications alone.

[0192] The above experimental results fully demonstrate that the drug carrier of the dual-response thermosensitive gel of the present invention has significantly stronger targeted drug delivery and on-demand drug release performance compared with HA or TK alone, thus improving the therapeutic effect of the drug. Under the premise of a constant number of modifiable sites on the MSN surface, the baseline efficacy of the single-modification system (HA or TK) was set as the reference standard (normalized value = 1). Experimental data show that the therapeutic effect of the TK alone modification system is significantly better than that of the HA alone modification system. It is worth noting that when HA and TK ligands are grafted onto the azido and amino sites on the MSN surface respectively (keeping the total modification amount constant), the therapeutic effect not only does not show competitive inhibition, but also exhibits a superadditive effect exceeding that of the TK single modification system. This phenomenon indicates that the HA / TK heteroligands, through spatial synergy, simultaneously enhance the active targeting affinity and microenvironment-responsive drug release kinetics of the nanocarrier, thereby achieving "synergistic gain" rather than simple functional superposition.

[0193] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a dual-response thermosensitive gel for drug carriers, characterized in that, The preparation method includes the following steps: Preparation of S1, N3 / NH2-MSN: Add MSNs to hybrid Aptex and A Z In xylene of PTES, after stirring thoroughly under nitrogen at room temperature for 24 hours, the product is centrifuged, washed three times with anhydrous ethanol, and then dried under vacuum at 50°C to obtain a white powder product, which is N3 / NH2-MSN. Preparation of S2 and TK-MSN: 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, EDC·HCl, and NHS were added to MES buffer at a molar ratio of 1:1.5~2:1~2. The mixture was stirred at room temperature under nitrogen atmosphere in the dark to induce an acylation reaction, yielding a ROS-responsive crosslinking agent. Then, a dispersed N3 / NH2-MSN solution was added dropwise, and the mixture was stirred at room temperature under nitrogen atmosphere in the dark for 24~48 hours. The precipitate was collected by centrifugation and washed with acetone and anhydrous ethanol. After washing, the product was vacuum dried to obtain a white precipitate, TK-MSN. Preparation of S3 and PEG-TK-MSN: Add EDC·HCl and NHS to the TK-MSN solution and activate the reaction for 1.5-3 hours; slowly add 10-20 mg / mL mPEG-NH2 solution, stir at room temperature under nitrogen and in the dark for 24-36 hours; collect the precipitate by centrifugation, wash with anhydrous ethanol and acetone alternately, freeze-dry under vacuum to obtain the product PEG-TK-MSN; Preparation of S4, PEG-HA & TK-MSN: EDC·HCl, NHS, and HA were dissolved in MES buffer and activated for 20-30 min. DBCO-NH2 was added to adjust the pH to 7.4, and the mixture was stirred overnight at room temperature. The reaction solution was then transferred to a dialysis bag and dialyzed in ultrapure water for 48 h. After lyophilization, HA-DBCO was obtained. This solution was added to a PEG-TK-MSN suspension and stirred at room temperature under nitrogen atmosphere in the dark for 24-36 h. The product was collected by centrifugation, washed 3-5 times with anhydrous ethanol, and then freeze-dried under vacuum to obtain PEG-HA&TK-MSN. S5. Preparation of thermosensitive gel: Add poloxamer P407, poloxamer P188, and inulin to a 10-20 mg / mL PEG-HA&TK-MSN solution, stir at room temperature, and after the poloxamer and inulin dissolve, let the mixed solution stand at 3-8℃ for 18-24 hours, then freeze at -20℃ for 2 hours and alternate between freeze-thaw at room temperature. Repeat this freeze-thaw cycle at least 3 times to obtain a dual-response thermosensitive gel.

2. The preparation method according to claim 1, characterized in that, In step S2 of the preparation method, the acylation reaction takes 0.5 to 3 hours.

3. The preparation method according to claim 1, characterized in that, In step S2 of the preparation method, the mass concentration of N3 / NH2-MSN is 5-10 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step S3 of the preparation method, the mass ratio of TK-MSN, EDC·HCl, NHS and mPEG-NH2 is 1:0.1~0.5:0.1~0.5:1~1.

5.

5. The preparation method according to claim 1, characterized in that, In step S4 of the preparation method, the mass ratio of EDC·HCl, NHS and HA is 1.5~2:0.8~1.2:1; the mass concentration of PEG-TK-MSN is 8-15 mg / mL.

6. The preparation method according to claim 1, characterized in that, In step S5 of the preparation method, the mass ratio of PEG-HA&TK-MSN, poloxamer P407, poloxamer P188 and inulin is 1~2:18~22:2~4:

2.

7. A dual-response thermosensitive gel for drug carrier obtained by the preparation method according to any one of claims 1 to 6.

8. The method for preparing curcumin-dual-responsive thermosensitive gel according to any one of claims 1 to 6, characterized in that, In step S1, N3 / NH2-MSN is replaced with N3 / NH2-MSN / Cur. The preparation method of N3 / NH2-MSN / Cur is as follows: N3 / NH2-MSN is added to an anhydrous ethanol solution of curcumin with a concentration of 4~10 mg / mL, pH 6.5~pH 7, stirred at room temperature in the dark for 24h~48h, centrifuged, washed with anhydrous ethanol, and then vacuum dried at room temperature for 8~12h. The resulting yellow precipitate is N3 / NH2-MSN / Cur.

9. Curcumin-dual-response thermosensitive gel obtained by the preparation method of claim 8.

10. The use of the curcumin-dual-response thermosensitive gel according to claim 9 in the preparation of drugs or health products for the prevention, relief and / or treatment of neurodegenerative diseases.

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