A Nose-to-Brain Delivery System for Taxifolin Ameliorates Alzheimer’s Disease via Synergistic Attenuation of Oxidative Stress and Mitochondrial Dysfunction

Abstract

The blood–brain barrier (BBB) presents the principal obstacle to drug delivery for Alzheimer’s disease (AD), severely restricting brain bioavailability and therapeutic efficacy. Taxifolin (TF) is a potent natural antioxidant with significant therapeutic potential. To enhance its efficacy in treating AD, we developed a brain-targeted delivery system based on a taxifolin-loaded thermosensitive hydrogel (TF-Gel). This platform integrates TF with a poly(N-isopropylacrylamide)-based thermosensitive hydrogel to enhance brain delivery, tissue penetration, and intracerebral retention via intranasal administration. TF-Gel exhibits excellent structural stability and functional performance, enabling efficient bypass of the BBB through the nasal–brain pathway. Furthermore, it regulates mitochondrial dysfunction, reverses abnormal levels of adenosine triphosphate (ATP), reactive oxygen species (ROS), and malondialdehyde (MDA) in neuronal mitochondria, repairs mitochondrial energy metabolism, restores mitochondrial dynamic balance, improves oxidative stress damage, and blocks cell apoptosis pathways. Collectively, these results highlight the strong potential of the TF-Gel nasal delivery system as a mitochondria-targeted therapeutic strategy for AD.

1. Introduction

Alzheimer’s disease (AD) is a major neurodegenerative disorder with significant global health implications, characterized by a gradual onset and progressive cognitive decline. Clinically, AD manifests as a spectrum of impairments, including memory loss, language deficits, and executive dysfunction [1,2]. Although historically regarded as an age-associated disease, AD is increasingly diagnosed in middle-aged populations. Projections suggest that by 2050, the global prevalence of AD will surpass 100 million [3,4]. Currently, pharmacological treatments for AD are mainly centered around acetylcholinesterase inhibitors, such as tacrine and rivastigmine [5,6]. While these therapies can slow disease progression and alleviate clinical symptoms, they are limited by factors such as narrow targeting, low selectivity, and significant side effects [7,8]. Moreover, despite extensive investigation into natural compounds with anti-AD potential, most existing therapies are administered orally and face substantial challenges in traversing the blood–brain barrier (BBB), thereby restricting their therapeutic efficacy and translational potential [9,10].

In recent years, nasal drug delivery systems have garnered significant attention for the treatment of brain diseases [11]. The primary advantage of nasal administration lies in the reduction in systemic drug exposure by bypassing hepatic first-pass metabolism and gastrointestinal degradation [12,13]. This route facilitates direct delivery of therapeutics to the central nervous system via the trigeminal and olfactory pathways, thereby bypassing the BBB [14]. Consequently, nasal delivery can minimize drug degradation, reduce systemic side effects, and enhance brain-targeted therapy with high drug loading and efficient delivery [15,16]. Therefore, the development of an optimized nasal drug delivery system holds great promise for enhancing the therapeutic efficacy of anti-Alzheimer’s agents.

Hydrogels, formed through the crosslinking of hydrophilic polymer chains into a three-dimensional (3D) network structure, are widely recognized for their high drug-loading capacity [17]. In particular, thermoresponsive hydrogels offer distinct advantages owing to their temperature-sensitive behavior. These hydrogels can be fabricated at ambient temperature, allowing for high-loaded drug incorporation, and exhibit a reversible lower critical solution temperature (LCST). At nasal cavity temperatures, the gel’s viscoelasticity increases, enhancing both its drug release rate and sustained release performance [18,19].

Various materials have been explored for thermoresponsive hydrogel fabrication. While alternatives such as poloxamers, chitosan/β-glycerophosphate systems, and methylcellulose have been investigated for nasal delivery, they often present certain limitations compared to PNIPAM. For instance, poloxamers may require high concentrations or additives to achieve suitable gelation at nasal temperature and can exhibit relatively weak gel strength [20]; chitosan-based systems are sensitive to pH and ionic strength, making gelation control less straightforward [21]; and methylcellulose typically undergoes a broad gelation range rather than a sharp phase transition [22]. In contrast, poly(N-isopropylacrylamide) (PNIPAM) is among the most extensively studied due to its sharp thermoresponsive hydrophilic–hydrophobic transition and reversible LCST behavior. These features make PNIPAM-based hydrogels particularly suitable for nasal drug delivery systems [23,24]. Notably, PNIPAM exhibits an LCST of approximately 32 °C, which closely matches the physiological temperature of the nasal cavity (33–35 °C) [25,26]. This temperature window enables the hydrogel to undergo a transition at the nasal temperature, increasing its viscoelasticity, which improves the adhesion to the nasal mucosa, reduces mucociliary clearance, and consequently enhances the sustained release of the drug in the nasal cavity. Moreover, PNIPAM hydrogels exhibit excellent biocompatibility and biodegradability [27,28], positioning them as an ideal drug delivery system for clinical applications.

Taxifolin (TF), also known as dihydroquercetin, is a flavonoid (specifically, a dihydroflavonol) with the molecular formula C₁₅H₁₂O₇. Its core structure consists of two benzene rings connected by a heterocyclic pyran ring with a carbonyl group at the C4 position and a dihydroxylated C ring [29]. It is commonly found in milk thistle, pine trees, and citrus fruits. It has been approved as a novel food and food additive in numerous countries worldwide [30]. The European Food Safety Authority has approved taxifolin as a safe food ingredient [31]. As a natural polyphenolic compound, taxifolin exhibits strong antioxidant capacity and a broad spectrum of biological activities, leading to its extensive use in the food, nutraceutical, and pharmaceutical sectors. Its broad-spectrum biological effects, particularly its potent antioxidant, anti-inflammatory, and neuroprotective activities, have attracted considerable research interest in the context of neurodegenerative diseases [32,33]. Accumulating evidence indicates that taxifolin exhibits remarkable antioxidant activity, anti-inflammatory effects, immune system modulation, vascular protection, and other pharmacological benefits [34,35]. Furthermore, emerging studies suggest its potential in enhancing cognitive function and mitigating the effects of aging [36,37]. Despite these promising findings, one limitation remains: the primary source of taxifolin is pine trees, has a long growth cycle and limited geographic distribution, resulting in low extraction yields and a relatively scarce resource base [38]. Consequently, the efficient utilization of taxifolin at low doses remains a critical challenge and constitutes a central focus of our ongoing research efforts. Although taxifolin has a wide range of biological activities, its poor water solubility and significant first-pass effect result in low oral bioavailability, severely limiting its clinical application [39]. To overcome this bottleneck, researchers have developed various novel delivery systems, such as liposomes, nanoparticles, and lipid nanocapsules, aimed at improving their solubility, stability, and in vivo delivery efficiency [40]. However, existing formulations still face challenges in achieving efficient nasal delivery and ensuring sufficient drug delivery to the central nervous system [41]. Therefore, this study aims to develop a new nasal drug delivery system based on a temperature-sensitive hydrogel. This system utilizes its temperature-sensitive in situ gel properties, which are expected to form a long-term retention in the nasal cavity, enhance mucosal adhesion, and reduce clearance, thus overcoming the limitations of existing preparations and providing a new strategy for achieving sustained and efficient nasal brain delivery of dihydroquercetin.

Building upon the research background outlined above and integrating prior findings from our research group, we developed a taxifolin-loaded thermosensitive hydrogel (TF-Gel) and systematically evaluated its properties following intranasal administration. Furthermore, we investigated the molecular mechanisms underlying the brain-targeted delivery and therapeutic effects of the TF-Gel in ameliorating cognitive deficits associated with AD. Based on the core exploration indicators of behavioral science, oxidative stress, and mitochondrial dysfunction, the required sample size for the experiment was calculated. This study aims to establish a mechanistic and theoretical framework supporting the potential of taxifolin-loaded thermosensitive hydrogels as a targeted therapeutic strategy for Alzheimer’s disease.

2. Materials and Methods

2.1. Materials

Taxifolin (purity > 98.0%), N-isopropylacrylamide (NIPAM), tetramethylethylenediamine (TEMED), potassium persulfate (KPS), and polyvinylpyrrolidone (PVP-K30) were all purchased from Shanghai McLean (Shanghai, China). Methacryloyl gelatin (GelMA, grafting rate of 90%) was purchased from Wenzhou Shuhe Biotechnology Co., Ltd. (Wenzhou, China). Deionized water was purchased from Solaibao (Beijing, China). The HT22 cell line (mouse hippocampal neuronal cells) was obtained from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China) (Catalog: CL-0697) and was cultured under standard conditions. The cell line was authenticated by STR profiling and tested negative for mycoplasma contamination; ATP, ROS, MDA assay kits were purchased from Shanghai Enzyme linked Biotechnology Co., Ltd. (Shanghai, China). The water was ultrapure water, located in the experimental building of Jilin Agricultural University (Jilin, China). The anti-Bax antibody, anti-Bcl-2 antibody, anti-Opa1 antibody, and anti-Drp1 antibody were purchased from Wuhan Saiwei Biotechnology Co., Ltd. (Wuhan, China). The anti-Mfn1 antibody, anti-Mfn2 antibody, anti-Fis1 antibody, and anti-Cyto-c antibody were purchased from Jiangsu Qinke Biological Research Center Co., Ltd. (Changzhou, China). The anti-cleaved caspase-3 antibody and anti-cleaved caspase-9 antibody were purchased from Aibo Anti (Shanghai) Trading Co., Ltd. (Shanghai, China) All other reagents were of analytical grade and could be obtained from commercial sources.

2.2. Preparation of TF-Gel

The taxifolin temperature-sensitive hydrogel was prepared by the cold solution method [42]. The specific operation is as follows: 1.02 g of NIPAM was ground into powder, dissolved in 3 mL of deionized water, 1.5 mL of 0.5 wt% GelMA was added, and the mixture was stirred for 20 min. Add 1 mL of 2 wt% KPS solution and 1 mL of TEMED in sequence, immediately place in an ice-water bath, and stir for 10 min to obtain a homogeneous precursor preparation (PNIPAM). Based on the effective dose of paclitaxel obtained from previous laboratory work, accurately weigh 4.0 mg of taxifolin and 0.6 mg of PVP-K30, dissolve them together in 1.0 mL of deionized water, and stir thoroughly to obtain a uniform drug solution. Subsequently, 1.0 mL of PNIPAM precursor prepared by the above method was measured, and the drug solution was slowly injected into it under continuous stirring, thoroughly mixed, and homogenized. In the final obtained hydrogel, the concentration of taxifolin was 0.4% (w/v). All the prepared hydrogels were stored at 4 °C for 24 h to make them fully swollen for standby. The taxifolin hydrogel was centrifuged at 12,000 r/min and 20 °C for 10 min to remove bubbles to obtain the taxifolin hydrogel. At the same time, the blank hydrogel without taxifolin was prepared.

2.3. Phase Transition of TF-Gel Sol-Gel

To assess the temperature sensitivity of the prepared hydrogel and determine the LCST of taxifolin hydrogel, a tube inversion method was employed. The taxifolin hydrogel was placed in a water bath, where the temperature was gradually increased from 10 °C to 50 °C at a rate of 1 °C/min. The gelation temperature was recorded as the temperature at which the solution completely lost its fluidity. The gelation time of both the blank hydrogel and the taxifolin hydrogel was then measured at this gelation temperature. All hydrogels were freshly prepared and stored at 4 °C for future use.

2.4. TF-Gel Structure Characterization

2.4.1. Scanning Electron Microscope (SEM)

The hydrogel samples were freeze-dried and examined by scanning electron microscopy (SEM) to characterize their surface microstructure. Fix the sample on the console, spray gold, scan the gold-sprayed sample under current, and record the appearance image of the magnified lens under 1000× and 2000× conditions.

2.4.2. Fourier Transform Infrared Spectroscopy (FT-IR) Detection

The freeze-dried test sample was mixed, ground, and pressed into a thin sheet by using the potassium bromide particle method under the infrared lamp. The scanning range was 500–4000 cm⁻¹ by using the Fourier transform spectrometer. The prepared hydrogel was subjected to an FT-IR spectrum to confirm the composition of the taxifolin hydrogel.

2.4.3. TF-Gel Particle Size Detection

The particle size of taxifolin thermosensitive hydrogel was determined by dynamic light scattering (DLS). Dilute taxifolin thermosensitive hydrogel to 1.0 mg/mL with deionized water and determine its hydrodynamic diameter and distribution.

2.4.4. TF-Gel Thermogravimetric Analysis

The thermal properties of the taxifolin-loaded thermosensitive hydrogel were evaluated by thermogravimetric analysis. Take 5 mg of both the taxifolin-loaded hydrogel and the blank hydrogel, place them in a thermogravimetric analyzer, and measure the quality change in hydrogel under the temperature range of 20–900 °C in the nitrogen environment at a heating rate of 10 °C/min; establish a relationship diagram between weight (%) and temperature (°C), and analyze the thermal decomposition of the hydrogel.

2.5. TF-Gel Rheological Evaluation

2.5.1. Temperature Scanning

Rheological studies were conducted using a Haake Mars60 rheometer (Thermo Fisher Scientific, Waltham, MA, USA). Heat the sample from 15 °C to 40 °C at a rate of 2 °C/min and measure the changes in elastic modulus (G′) and viscosity modulus (G″) with temperature to detect temperature scanning.

2.5.2. Frequency Sweep

At 25 °C, with a fixed strain value of 1%, frequency scanning is performed within the range of 0.1–100 rad/s to detect the changes in G′ and G″ with frequency.

2.5.3. Shear Viscosity

In order to evaluate the relationship between the viscosity of hydrogel and the shear rate, the shear rate was increased from 0.1/s to 100/s at 25 °C.

2.5.4. Stress–Strain Scanning and Strain–Time Scanning

At 25 °C, the G′ and G″ of the hydrogel under different strain conditions were measured, γ = 0.1–100%, and the frequency was fixed at 1 Hz. The test hydrogel was cycled several times under the conditions of small and large oscillating strains, and the G′ and G″ were measured. The scanning conditions were switched from γ = 1% and γ = 200%, respectively. The strain interval was 120 s, and the number of cycles was 5.

2.6. In Vitro Nasal Mucosal Release Study of TF-Gel

A Franz diffusion cell was used to evaluate the osmotic release of the taxifolin-loaded thermosensitive hydrogel. Fix the pig nasal mucosa sample between the donor and recipient compartments, with the front and back of the mucosa facing the recipient and donor compartments, respectively. Both compartments are filled with artificial, simulated, synthetic nasal mucus (BZ251), and the recipient compartment is continuously stirred at a speed of 200 r/min and maintained at a constant temperature of 34 ± 1.0 °C. At specific times (0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), 1 mL of simulated nasal mucus is taken out, and an equal amount of fresh simulated nasal mucus is added to fill. The concentration of taxifolin in the collected release medium was quantified by high-performance liquid chromatography (HPLC). The analysis was performed using a 1260 Agilent high-performance liquid chromatograph with an Eclipse XDB-C18 chromatographic column (4.6 ID × 250 mm). The mobile phase consisted of a mixture of methanol and 0.1% phosphoric acid in water (volume ratio 39:61). Chromatographic conditions were as follows: column temperature, 35 °C; volume flow rate, 1.0 mL/min; detection wavelength, 291 nm; injection volume, 10 μ L. Under these conditions, taxifolin showed a retention time of approximately 7 min.

2.7. TF-Gel Security Evaluation

2.7.1. Cytocompatibility

The CCK-8 method was used to detect the effect of taxifolin thermosensitive hydrogel on the activity of HT22 cells. The operation steps for the CCK-8 method experiment are as follows: HT22 cells are added to a 96-well plate (5 × 10³/well) containing 10% DMEM medium, and cultured in a CO₂ incubator for 24 h. After the blank hydrogel and taxifolin hydrogel are sterilized under ultraviolet light for 2 h, the original culture medium is replaced with the culture medium containing control hydrogel and taxifolin hydrogel, respectively, and the culturing continues for 24 h. Remove the culture medium, add 10 μL CCK-8 to each well, and incubate for 4 h. Measure the absorbance at 450 nm, and calculate the cell vitality according to the following formula:

Cell viability (%) = (A_s − A₀)/(A_c − A₀) × 100%

(1)

Here, A_s is the absorbance of the sample, A₀ is the absorbance of the zeroing well, and A_C is the absorbance of the control group.

2.7.2. Blood Compatibility

Take fresh blood from SPF-grade ICR mice. The mice were provided by Changchun Yisi Experimental Animal Technology Co., Ltd. (Changchun, China). Collect whole blood and place it in a centrifuge tube soaked in heparin sodium. Centrifuge at 5000 r/min and 4 °C for 10 min, remove the supernatant, add an appropriate amount of PBS solution, slowly wash and shake well, centrifuge again for 10 min, further wash the red blood cells, repeat the above steps three times until the upper clear liquid is clear and transparent, add PBS to dilute to the final concentration of 2% red blood cell suspension. Take a 2 mL centrifuge tube, add 1 mL of hydrogel soaked in PBS solution, then add 0.5 mL of prepared red blood cell suspension, incubate at 37 °C for 2 h, centrifuge at 2000 r/min, 4 °C for 5 min, absorb 100 μL of supernatant and add it to 96 well plate, use a multi-function microplate meter to measure the absorbance at 545 nm. An equal amount of deionized water was used as the positive control group, and PBS solution was used as the negative control group. Calculate the hemolysis rate according to the following formula:

Hemolysis rate (%) = (Sample absorbance − Negative control absorbance)/(Positive control absorbance − Negative control absorbance) × 100%

(2)

2.7.3. Weight Testing

ICR mice (half male and half female) were randomly divided into three groups: control group, intranasal administration of Gel group, and intranasal administration of TF-Gel group, with 10 mice in each group. The maximum dose of intranasal administration was administered once a day for 14 days, and the mice were weighed every 3 days.

2.7.4. Organ Tissue Pathological Examination

The mice were euthanized using the neck removal method. The nasal mucosa, brain tissue, heart, liver, spleen, lungs, and kidneys of the mice were isolated and fixed in 4% paraformaldehyde solution. After embedding in paraffin, the slices were cut into 5 μm-thick sections for histopathological analysis using the H&E staining method. The histopathological identification of each organ tissue was performed using the Olympus BX51 optical microscope (Olympus, Tokyo, Japan).

2.8. TG-Gel Pharmacokinetic Testing

ICR mice (18–20 g) were adaptively housed in a standard laboratory environment (25 ± 2 °C, 50 ± 5% relative humidity, 12 h light/dark cycle) for one week, during which they had free access to water and food. Randomly divide mice into a nasal administration group (IN) and a tail vein administration group (IV). For the pharmacokinetic studies, the number of mice at each sample collection time point was five. TF-Gel (40 mg/kg) was administered to 0.5 μL/g of the nasal cavity, and taxifolin solution (40 mg/kg) was administered to the tail vein in a dose of 0.1 mL/10 g. Blood samples were collected from the eyeballs at fixed time points (5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h after administration), immediately stored at 20 °C, and centrifuged at 3000 r/min for 10 min; plasma was collected and stored at −80 °C for testing.

After blood collection, the mice were euthanized using the neck removal method. The mice were placed on an ice table, and their brain tissue was quickly dissected and removed. The tissue was then washed with physiological saline to remove all adherent fluids.

Determine the concentration of taxifolin in plasma and brain tissue by HPLC, and measure the maximum concentration (C_max) of taxifolin in plasma and brain, the time to reach the maximum concentration (T_max), the area under the curve (AUC), the clearance (CL), the half-life (T_1/2), the mean residence time (MRT), and plot the drug concentration time curve to establish the pharmacokinetic curve of taxifolin. Calculate drug targeting potential (DTP) and drug targeting efficiency (DTE); the calculation formula is as follows:

$$\mathrm{DTP} \% = {\displaystyle \frac{{\left({\mathrm{AUC}}_{\mathrm{Brain}}\right)}_{\mathrm{IN}}-{\left({\mathrm{AUC}}_{\mathrm{Brain}}\right)}_{\mathrm{IV}}{\left({\mathrm{AUC}}_{\mathrm{Blood}}\right)}_{\mathrm{IN}}/{\left({\mathrm{AUC}}_{\mathrm{Blood}}\right)}_{\mathrm{IV}}}{{\left({\mathrm{AUC}}_{\mathrm{Brain}}\right)}_{\mathrm{IN}}}} \times 100$$

(3)

$$\mathrm{DTE} \%={\displaystyle \frac{{[{\mathrm{AUC}}_{\mathrm{Brain}}/{\mathrm{AUC}}_{\mathrm{Blood}}]}^{\left(\mathrm{IN}\right)}}{{[{\mathrm{AUC}}_{\mathrm{Brain}}/{\mathrm{AUC}}_{\mathrm{Blood}}]}^{\left(\mathrm{IV}\right)}}} \times 100$$

(4)

2.9. TF-Gel In Vivo Brain Targeting Research

Prepare DiR-loaded tracer nanoparticles (DiR-PLGA-NP) using the optimal preparation method and measure their particle size. Inject 0.2 mL/10 g into the nasal cavity of mice while setting up a control group of taxifolin by gavage. At different time points after injection (1, 3, 6, 12, 24, 48 h), observe and take pictures of the head and whole body of the experimental animals using a small-animal live fluorescence imaging device. Each animal underwent X-ray and fluorescence imaging. Carestream MI software (version 5.4) was used to overlay and locate the X-ray and fluorescence images to study the distribution of tracer nanoparticles in different tissues of mice.

2.10. The Therapeutic Effect of TF-Gel on Okadaic Acid (OA)-Induced AD Mice

2.10.1. Establishment of OA-Induced AD Mouse Model

A total of 40 healthy male ICR mice, weighing 18–20 g, were obtained from Changchun Yisi Experimental Animal Technology Co., Ltd. (certificate number: SCXK-2020-0001). After one week of acclimatization under controlled conditions (22 ± 2 °C, 60 ± 5% humidity, alternating 12 h light/dark cycles), using a random allocation method, mice were assigned to four experimental groups in a single order: blank control group (Control), model group (AD), nasal administration TF-Gel group (TF-Gel), and nasal administration Gel group (Gel). The individual animal was considered the experimental unit for all interventions and statistical analyses. The sample size (n = 10) refers to the number of animals per group. The sample size is estimated based on the size of the observed effects in our preliminary experiments. Using the same statistical parameters as the research content, estimate the required sample size for each group to be n = 8–10. Therefore, we set n = 10 to ensure sufficient power. To ensure complete anesthesia, all mice were intraperitoneally administered 0.2 mL/20 g of 0.4% pentobarbital sodium. Following anesthesia, the mice were secured on a stereotaxic apparatus. After shaving and disinfecting the scalp, a midline incision was made to expose the skull. A small incision was made over the scalp, and the bilateral hippocampal CA1 region was targeted as the administration site. Using the anterior fontanelle as a reference, the coordinates were defined as 2 mm posterior to the lateral ventricle surface and 1.5 mm on either side of the sagittal suture. A miniature skull drill was used to create a small hole approximately 2.5 mm deep in the skull surface. A microinjector was then used to administer 2 μL of okadaic acid (10 μg/0.1 mL) at a constant injection rate of 0.2 μL/min. The needle was inserted at a speed of 1 mm/min, and, after injection, it was allowed to remain in place for 1 min before being slowly withdrawn at the same rate. In the control group, 2 μL of physiological saline was injected instead of OA. Following the procedure, the scalp incision was sutured, and penicillin was administered intraperitoneally for three consecutive days to prevent infection. The mice were then placed in a temperature-controlled incubator at 25 °C to maintain a stable body temperature.

After 3 days of postoperative observation, the physiological status of the mice was confirmed to be back to normal. After equivalent conversion based on pharmacokinetic and bioavailability results, the TF-Gel group received intranasal injections of TF-Gel at a dosage of 2 mg/kg/d. In order to exclude the potential non-specific effect of blank hydrogel on the AD model, a Gel group (blank hydrogel for nasal administration) was set up. Based on chronic characteristics of AD. Except for the model group, all other groups of mice were administered continuously for 60 days. The daily administration time was 10 am, and the medication was administered in the Control, Gel, and TF-Gel groups in a fixed sequence, with no change in the administering personnel. Animals were only included in the study if they completed the entire experimental process without complications. The specific exclusion criteria were as follows: postoperative mortality or severe health deterioration unrelated to treatment; technical malfunctions during drug administration or sample collection; and failure to meet the predefined disease model criteria (for example, the swimming time of mice in behavioral experiments after the experiment did not conform to the model rules).

Blinding was not implemented in this study. All personnel involved in the animal allocation, experimental procedures, outcome assessment, and data analysis were aware of the group identities.

2.10.2. Behavioral Testing

Five days before the end of treatment, Morris water maze (MWM) and computer video tracking system were used to measure, record, and evaluate the neural memory, cognition, and motor function of mice. Before formally recording the experimental results, it was necessary to train mice continuously for 5 days, with each mouse alternating 5 times a day. The hidden-platform training test was conducted over four consecutive days (days 1–4). After the training, behavioral video analysis software was used to conduct localization navigation experiments and spatial probe tests on mice for 180 s. The time it took mice to find an underwater platform was used as the evaluation criterion.

2.10.3. Mitochondrial Function Testing

Hippocampal tissues were rapidly harvested, homogenized in ice-cold 0.9% saline, and centrifuged at 1000× g for 10 min at 4 °C. Transfer the supernatant to a test tube for subsequent analysis, and evaluate the levels of ATP, ROS, and MDA using goat anti-mouse ATP, ROS, and MDA ELISA kits according to the provided instructions.

Western blot analysis was used to detect the protein expression of cytochrome C (Cyto-C), Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, mitochondrial fusion proteins mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (Opa1), mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1) in hippocampal tissue.

2.11. Statistical Processing

Statistical analysis was conducted in the form of mean ± standard deviation (SD). Prism 8.0.2 software (GraphPad Software, Boston, MA, USA) and Origin 2024 software were used to represent data analysis. A t-test was used to analyze data. When p < 0.05, p < 0.01, or p < 0.001, the difference was considered statistically significant. Otherwise, it was considered to be insignificant.

3. Results

3.1. LCST

The synthesized hydrogel is transparent, homogeneous, and maintains a flowable liquid state at lower temperatures. Upon reaching a temperature range of 33–34 °C, a gelation process is initiated. After gelation, the hydrogel transitions into a milky white, non-flowing semi-solid. The gelation time, defined as the period required for the transformation from liquid to gel, is approximately 40–50 s (Figure S1).

3.2. Gel Characterization

3.2.1. SEM Analysis of the Hydrogel

SEM images (Figure 1A) reveal that the hydrogel exhibits a three-dimensional porous network with relatively uniform pore distribution and minor local heterogeneity. The pores were predominantly circular or elliptical, and incomplete “tentacle-like” structures were observed in some gaps, likely representing the termini of partially cross-linked polymer chains. Upon incorporation of taxifolin, the microstructural regularity of the hydrogel was markedly enhanced. The pore arrangement became highly ordered, and the “tentacle-like” structures largely disappeared. Instead, the hydrogel displayed regular square-shaped pores with smooth walls and more continuous, well-connected networks. These observations suggest that taxifolin in TF-Gel may interact with polymer chains to form hydrogen bonds, thereby improving the material properties of the hydrogel.

3.2.2. FT-IR Image of Gel

The FT-IR spectra of the Gel (Figure 1B) and TF-Gel (Figure 1C) reveal distinct features that confirm the successful incorporation of taxifolin into the hydrogel matrix. In the Gel, characteristic absorption bands corresponding to the C=O stretching vibration at 1659 cm⁻¹, and the N-H bending and C-N stretching vibrations at 1551 cm⁻¹, verify the presence of amide groups. Following taxifolin loading, a broad O-H stretching vibration appears at 3437.79 cm⁻¹, which is absent in the Gel, indicating the presence of the phenolic hydroxyl groups from taxifolin. The slight red shift in this peak from the typical 3500 cm⁻¹ position suggests the formation of hydrogen bonds between the phenolic hydroxyl groups of taxifolin and the amide groups of the hydrogel network. The broad and intense nature of the O-H peak in TF-Gel further supports this hydrogen-bonding interaction. Moreover, the C=O stretching vibration of the Gel shifts from 1659 cm⁻¹ to 1648.08 cm⁻¹ after taxifolin incorporation, consistent with the expected low-frequency shift caused by hydrogen bond formation. Additionally, the aromatic C=C vibrations of taxifolin contribute to the enhanced peak intensity at 1648.08 cm⁻¹, indicating effective blending of taxifolin with the hydrogel through hydrogen bonding or π–π stacking, without observable phase separation. Collectively, these results demonstrate that taxifolin modulates the hydrogel structure through noncovalent interactions, improving network stability and compatibility while preserving structural integrity and favorably supporting drug release performance in TF-Gel.

3.2.3. Gel Particle Size Detection

Figure 1C presents the particle size distribution of the gels measured by dynamic light scattering (DLS). The TF-Gel exhibits a normal distribution, with an average hydrodynamic diameter of 105.05 nm. This size range is favorable for adhesion to the nasal mucus layer, reducing rapid mucociliary clearance while minimizing the risk of airway obstruction. Furthermore, particles of this size can form a stable three-dimensional network structure upon gelling at physiological temperature, thereby limiting rapid drug diffusion. This property extends the local residence time of the drug, thereby enhancing its targeted delivery and therapeutic efficacy. The low polydispersity index (PDI) of 0.168 indicates a narrow size distribution, suggesting that the taxifolin is well-incorporated in an aggregated state, and the particle system demonstrates high uniformity and stability.

3.2.4. Thermogravimetric Analysis (TGA)

TGA results (Figure S2) reveal that the initial melting and decomposition temperature of pure hydrogel is approximately 310 °C, while the TF-Gel composite hydrogel containing taxifolin exhibits a significant mass loss at around 370 °C. This data suggests that the incorporation of taxifolin not only preserves the inherent thermal stability of the Gel but also notably delays the thermal decomposition process. This improvement is likely due to the increased cross-linking density of the 3D network structure, which enhances the overall thermal stability of the composite material.

3.3. Gel Rheological Evaluation

3.3.1. Temperature Scanning

As illustrated in Figure 2a, when the temperature of TF-Gel reaches 33 °C, both the elastic modulus (G′) and the viscous modulus (G″) increase sharply, indicating a sol-Gel phase transition at this temperature. This transition further confirms that the LCST value is 33 °C. At this point, both G′ and G″ values increase synchronously, suggesting that TF-Gel maintains a stable semi-solid state during the phase transition, exhibiting both viscous and elastic characteristics. Such a viscoelastic balance is critical for achieving intimate contact with the nasal mucosa and prolonging drug residence. As the temperature rises from 33 to 37 °C, both G′ and G″ continue to rise, signifying an enhancement in the mechanical strength of TF-Gel at physiological temperature compared to room temperature. This observation confirms that the mechanical properties of TF-Gel are optimized for the nasal environment, which not only aids in prolonging drug retention but also effectively resists mucociliary clearance, thus preserving the structural integrity and stability of the Gel.

3.3.2. Frequency Scanning

Figure 2b presents the dynamic frequency scanning analysis curve, illustrating the relationship between the storage modulus (G′) and the loss modulus (G″) as functions of angular frequency. At higher frequencies, the molecular interactions within the hydrogel become more pronounced, leading to an increase in both G′ and G″ with increasing angular frequency. This trend indicates that the hydrogel’s resistance to external forces strengthens as the angular frequency increases. As shown in the figure, both the G′ and the G″ of Gel and TF-Gel exhibit a positive correlation with angular frequency. Notably, the G′, which reflects the elastic properties, consistently exceeds the G″, which corresponds to the viscous behavior, thus confirming the gel-like characteristics of the material. Moreover, TF-Gel generally shows higher moduli compared to Gel. This suggests that the incorporation of taxifolin significantly enhances the cross-linking density of the hydrogel network, thereby improving its mechanical strength.

3.3.3. Shear Viscosity

Figure 2c illustrates the dependence of TF-Gel viscosity on shear rate, highlighting the shear-thinning behavior of the hydrogel. The viscosity of TF-Gel decreases as the shear rate increases, indicating its capacity for shear-thinning. This behavior is critical, as it suggests that TF-Gel possesses favorable 3D printability and material injectability.

3.3.4. Stress–Strain Scanning

The stress–strain scanning results (Figure 2d) indicate that, with increasing strain, the elastic modulus (G′) and viscous modulus (G″) of the Gel decrease significantly, and the Gel cross-linking network is unable to maintain its original structure. In contrast, the G′ and G″ of TF-Gel exhibit minimal changes as strain increases. This suggests that the deformation resistance of the hydrogel is notably enhanced upon incorporation of taxifolin. This improvement in mechanical stability is likely attributable to hydrogen bond interactions between the hydroxyl groups of taxifolin and the polymer chains, thereby increasing the rigidity of the hydrogel network.

3.3.5. Strain–Time Scanning

The strain–time scanning results for TF-Gel (Figure 2e) reveal that under low strain, the elastic modulus (G′) was dominant, indicating that the hydrogel network was tightly structured, exhibiting significant elastic response, and was in a swollen state. Concurrently, the viscous modulus (G″) remains relatively high, suggesting that the hydrogel retains some fluidity despite its overall rigidity. Under high strain, G′ decreases substantially, resulting in a reduced elasticity, whereas G″ exhibits only a slight decrease and becomes dominant, with its amplitude of change being significantly smaller than that of G′, which implies that the hydrogel viscosity dissipation capacity is less affected by strain. Upon repeating the entire process five times, the trend of modulus change after each strain cycle remains consistent. This indicates that the material maintains stable performance under repeated strain loading, demonstrating the hydrogel’s resilience to cyclic loading and excellent mechanical reversibility. As the measurements were conducted at a constant temperature of 25 °C, the observed rheological behavior was governed primarily by strain amplitude rather than thermal effects.

3.4. TF-Gel In Vitro Nasal Mucosal Release

To evaluate the release performance of TF-Gel, an in vitro nasal mucosa penetration study was conducted. The results, as depicted in Figure S3, demonstrate that TF-Gel exhibits a high dissolution rate within the first 12 h, characterized by rapid initial penetration. Over a 72 h-period, TF-Gel undergoes sustained and gradual release, with nearly 100% cumulative release observed, indicating a prolonged, slow release in the later stages. These findings suggest that the hydrogel 3D network structure, formed by incorporating taxifolin, plays a crucial role in facilitating an efficient and stable drug release. Overall, TF-Gel prolongs drug residence time while maintaining controlled, sustained release with high efficiency.

3.5. Safety Evaluation

3.5.1. Cell Compatibility

The cytocompatibility of Gel and TF-Gel toward HT22 cells was evaluated using the CCK-8 assay. As shown in Figure 3a, neither Gel nor TF-Gel exhibited detectable cytotoxicity, and the cell viability remained high. No significant difference in cell viability was observed between the two treatment groups, suggesting that the incorporation of taxifolin did not induce additional toxicity. Furthermore, these results may indicate a potential enhancement in cell proliferation, implying that the formulation possesses excellent cellular compatibility.

3.5.2. Blood Compatibility

As illustrated in Figure 3b, both Gel and TF-Gel exhibited hemolysis rates below 5%, meeting the safety standards for nasal administration. Notably, the hemolysis rate of TF-Gel was significantly lower than that of Gel (p < 0.01), suggesting that the incorporation of taxifolin improves the hydrogel biocompatibility.

3.5.3. Weight Monitoring

Figure 3c illustrates the changes in body weight of mice throughout the experiment. On the first day following administration, a slight reduction in body weight was observed, after which the weight showed a consistent upward trend. Compared to the blank group, the body weight of mice in both treatment groups remained slightly lower; however, the difference between the groups was not statistically significant (p > 0.05).

3.5.4. Pathological Analysis of Organ Tissues

Figure 3d presents the histological evaluation of the hydrogel on major tissues and organs in mice, assessed using HE staining. The tissues examined included the brain, nasal mucosa, heart, liver, spleen, lungs, and kidneys. No signs of inflammation or tissue damage were observed in any of the treatment groups, and no abnormalities were detected in the liver or kidney tissues. These findings indicate that neither Gel nor TF-Gel poses a risk of organ rejection or tissue dysfunction, demonstrating favorable biocompatibility and safety of TF-Gel within the intranasal injection sustained-release system, highlighting its potential therapeutic value for treating AD.

3.6. Pharmacokinetic Analysis

In the plasma pharmacokinetic analysis (Figure 4a), the concentration of taxifolin in the plasma of mice in the intravenous injection group exhibited a rapid decline over time, with detectable levels lasting only up to 8 h. In contrast, following intranasal administration of TF-Gel, the detection window for taxifolin in the plasma was extended to 24 h, and the drug release profile demonstrated a sustained-release effect. In the brain tissue pharmacokinetic analysis (Figure 4b), the concentration of taxifolin in the brain of mice in the intravenous injection group was minimal, with undetectable levels after 12 h of administration, indicating limited brain penetration due to the BBB. However, after intranasal administration of TF-Gel, the concentration of taxifolin in the brain tissue was significantly higher, with detectable levels present even after 48 h. These results indicate that TF-Gel can effectively control the slow, sustained release of the drug through the gel network swelling mechanism. Compared to intravenous injection, intranasal administration of TF-Gel not only bypasses the BBB but also leverages the hydrogel adhesive properties to prolong drug retention in the nasal cavity, thereby significantly enhancing drug delivery to the brain.

To further investigate the brain-targeting potential of TF-Gel, relevant pharmacokinetic parameters were calculated using the pharmacokinetic software DAS 2.0, as summarized in

Table 1. Plasma and brain pharmacokinetic parameters of TF intravenous injection and TF-Gel intranasal administration (n = 5).

Parameters	Intravenous Administration	Intranasal Administration
Plasma
T1/2 (h)	2.50 ± 0.34	15.98 ± 3.79 **
Tmax (h)	0.11 ± 0.05	1.08 ± 0.42 **
Cmax (mg/L)	15.35 ± 2.87	7.47 ± 1.65 *
AUC0-t (ng/mL * h)	18.58 ± 1.25	36.87 ± 4.60 *
MRT0-t (h)	2.16 ± 0.24	6.16 ± 1.21 **
CL(L/h/kg)	1.62 ± 0.54	1.09 ± 0.25
Brain
T1/2 (h)	2.27 ± 0.68	22.96 ± 2.76 **
Tmax (h)	0.51 ± 0.32	3.87 ± 0.81 *
Cmax (mg/L)	7.87 ± 1.25	38.27 ± 4.44 **
AUC0-t (ng/mL * h)	17.87 ± 4.41	883.99 ± 29.47 **
MRT0-t (h)	2.21 ± 0.12	17.09 ± 1.78 **
CL(L/h/kg)	2.14 ± 0.60	0.04 ± 0.01 **
Brain/plasma ratio
DTE	2492.85%
DTP	95.99%

* p < 0.05, ** p < 0.01 vs. intravenous administration ($\overline{x}$ ± s, n = 5).

. In plasma, the half-life (T_1/2) and mean residence time (MRT_0-t) following intravenous (IV) administration were 2.50 ± 0.34 h and 2.16 ± 0.24 h, respectively, indicating rapid drug elimination from the plasma. In contrast, the T_1/2 (15.98 ± 3.79 h) and MRT_0-t (6.16 ± 1.21 h) following intranasal (IN) administration were significantly prolonged, approximately 6.3 times and 2.85 times longer than those observed with IV administration. Additionally, the T_max for IN (1.08 ± 0.42 h) was markedly higher than for IV (0.11 ± 0.05 h). Although the maximum concentration (C_max) of IN (7.47 ± 1.65 mg/L) was significantly lower than that of IV (15.35 ± 2.87 mg/L), the area under the concentration-time curve (AUC_0-t) for IN (36.87 ± 4.60 ng/mL * h) was higher than that of IV (18.58 ± 1.25 ng/mL * h). The prolonged T_1/2 and MRT_0-t in the IN suggest slower drug release into plasma, which leads to reduced systemic exposure while facilitating prolonged drug delivery to the brain. The clearance (CL) of IN (1.09 ± 0.25 L/h/kg) was lower than that of IV (1.62 ± 0.54 L/h/kg), further confirming a slower drug elimination rate in the IN. In brain tissue, the AUC_0-t for IN (883.99 ± 29.47 ng/g * h) was 49.5 times higher than that for IV (17.87 ± 4.41 ng/g * h), and the C_max for IN (38.27 ± 4.44 mg/g) was 4.9 times greater than that for IV (7.87 ± 1.25 mg/g). These results demonstrate that IV administration has extremely low efficiency in crossing the BBB, leading to limited drug exposure in the brain. In contrast, nasal administration of TF-Gel significantly enhances drug exposure in the brain. The T_1/2 for IN brain tissue (22.96 ± 2.76 h) was 10.1 times that of IV (2.21 ± 0.12 h), while T_max (3.87 ± 0.81 h) and MRT_0-t (17.09 ± 1.78 h) were 7.5 and 7.7 times higher, respectively, compared to IV (0.51 ± 0.32 h and 2.21 ± 0.12 h). These findings suggest that TF-Gel, administered via the nasal route, bypasses the BBB and achieves direct brain targeting via the nasal mucosa–brain pathway. Moreover, the CL for IN in brain tissue (0.04 ± 0.01 L/h/kg) was significantly lower than that for IV (2.14 ± 0.06 L/h/kg), indicating a prolonged drug retention time and sustained therapeutic effect in the brain.

The high drug targeting efficiency (DTE, 2492.85%) and drug targeting percentage (DTP, 95.99%) further indicate that taxifolin can effectively target brain tissue following nasal administration via the olfactory or trigeminal pathways. This direct targeting bypasses the BBB and the systemic circulation, thereby mitigating the limitations associated with these routes.

3.7. Brain Targeted In Vivo Imaging

As depicted in Figure 5, compared to the intragastric administration group (IG), the drug fluorescence distribution in the nasal administration group (IN) is primarily concentrated in the brain, with a significantly faster distribution rate. Fluorescence signals were detectable within 1 h post-administration and remained observable up to 48 h, suggesting that the drug has high selective retention in the brain tissue. In contrast, the first detection of the drug fluorescence signal in the IG was observed in the abdominal region, with a delayed detection of brain fluorescence occurring at 3 h. Furthermore, the fluorescence intensity in the brain was markedly weaker in the IG compared to the IN. This disparity likely results from the need for the drug to cross the BBB via systemic circulation in the IG, whereas in the IN, the drug bypasses the BBB and enters the cerebrospinal fluid (CSF) circulation directly through the olfactory and trigeminal nerve pathways. This direct delivery mechanism allows the drug to circumvent the BBB, which is effectively avoiding the barrier effect of the BBB. Additionally, the systemic exposure of the IG to the drug likely diminishes its efficacy in the brain. In contrast, nasal administration of TF-Gel effectively mitigates this limitation, highlighting the superior brain-targeting efficiency of TF-Gel delivered via the nasal route.

3.8. The Mechanism of TF-Gel Anti-AD Effect

3.8.1. Behavioral Experiment

Although pre-defined inclusion/exclusion criteria were established (see Section 2.10.1), no animals, experimental units, or data points were excluded from the analysis in any of the experimental groups. All animals completed the entire study protocol, and all collected data were analyzed. Using the Morris water maze, thermal infrared imaging technology was employed to record the spatial exploration behaviors of mice within a defined time window (Figure 6A). The results indicated that mice in the control group demonstrated strong spatial learning capabilities, with their movement trajectories exhibiting purposeful and goal-directed characteristics. These mice were able to quickly locate the target quadrant containing the submerged platform, suggesting intact spatial memory function. In contrast, mice in the AD group displayed significant spatial cognitive impairments, as evidenced by their movement trajectories, which showed random, non-directed patterns. After treatment, the TF-Gel group showed significant improvement in spatial memory, while this improvement was not reflected in the Gel group. The observed cognitive impairment was directly evidenced by deficits in the Morris water maze, encompassing both the hidden-platform training (Figure 6B) and the subsequent spatial probe test (Figure 6C).

3.8.2. ELISA Evaluation of Mitochondrial Functional Parameters

To assess the protective effect of TF-Gel on mitochondrial function in the brains of AD mice, we evaluated changes in ATP, ROS, and MDA levels in the hippocampus across the different experimental groups. As shown in Figure 6D, compared to the control group, mice in the AD group exhibited a significant reduction in ATP levels (p < 0.01), alongside a notable increase in ROS and MDA levels (p < 0.01). In contrast, TF-Gel treatment groups demonstrated a significant reversal of mitochondrial dysfunction induced by Okadaic acid in the hippocampus of AD mice (p < 0.05, p < 0.01). Treatment with TF-Gel restored ATP expression, reduced ROS levels, and normalized MDA levels, thereby significantly mitigating the neurotoxic effects induced by Okadaic acid. Similarly, the Gel group did not show any significant effects in these aspects. These findings suggest that TF-Gel exerts a considerable therapeutic effect in improving mitochondrial dysfunction in AD mice.

3.8.3. Western Blot Analysis Results

WB analysis was conducted to evaluate protein factors related to mitochondrial structure and function, as well as apoptosis-related indicators, as shown in Figure 6E. Compared to the control group, the AD group exhibited significant upregulation of apoptosis markers, including cleaved caspase-3, cleaved caspase-9, and pro-apoptotic proteins such as Bax and Cyto-C, alongside a notable reduction in the anti-apoptotic protein Bcl-2. Following treatment with TF-Gel, these abnormalities were markedly ameliorated. Additionally, WB was used to assess proteins involved in mitochondrial dynamics, including the fusion proteins Mfn1, Mfn2, and Opa1. In the AD group, levels of the fission-related proteins Drp1 and Fis1 were significantly elevated, while the fusion proteins Mfn1, Mfn2, and Opa1 were markedly decreased. These findings suggest that mitochondrial dynamics in AD mice, induced by Okadaic acid, were severely impaired, resulting in an imbalance between mitochondrial fission and fusion processes and abnormal expression of mitochondrial dynamic proteins. TF-Gel treatment effectively normalized this imbalance by upregulating Mfn1, Mfn2, and Opa1 while suppressing Drp1 and Fis1 expression, thereby restoring mitochondrial dynamic homeostasis and preserving mitochondrial integrity.

4. Discussion

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that requires long-term, standardized pharmacotherapy [43]. However, due to the progressive nature of AD, patients often face significant challenges in maintaining consistent medication behavior, which imposes a substantial burden on both patients and their families. Therefore, the development of drug formulations with efficient controlled-release capabilities is crucial for improving patient adherence to treatment. In recent years, the nasal brain delivery system has garnered considerable attention in the treatment of neurodegenerative diseases, owing to its ability to bypass first-pass metabolism, overcome the blood–brain barrier, and enable rapid absorption with sustained drug release [44].

Taxifolin, a promising medicinal and edible compound, exhibits potent antioxidant activity [45]. Preliminary studies have shown that taxifolin significantly alleviates symptoms associated with AD. However, its limited availability remains a critical challenge [46]. Thus, a key research focus has been to explore ways to achieve long-term, efficient pharmacological effects of taxifolin while minimizing dosage. To address this, our research has concentrated on the development of a mild, sustained-release hydrogel-based delivery system for taxifolin [47].

Nasal administration, as a direct and non-invasive ‘nose brain’ delivery route, has shown unique advantages in the treatment of central nervous system diseases, allowing drugs to bypass the blood–brain barrier and increase brain drug concentration. However, this pathway also faces inherent challenges such as rapid clearance of nasal mucus and limited mucosal permeability [48]. In order to overcome these obstacles, researchers have developed a variety of strategies, including nanotechnology-based delivery systems and in situ gel systems [49,50]. Among them, thermosensitive in situ hydrogel has attracted much attention due to its unique solution gel transition characteristics: it exists in the form of a low viscosity solution at room temperature for easy administration, and rapidly converts into gel at nasal cavity temperature, which can extend the retention time of mucosa and control drug release. In this context, our study designed and constructed a poly(N-isopropylacrylamide) (PNIPAM)-based thermosensitive hydrogel system specifically for the nasal delivery of taxifolin. This innovative system diverges from previously reported nanocomposite gel formulations in both material selection and construction strategy [51]. We conducted a systematic investigation into various aspects of the hydrogel, including its sustained release performance, brain targeting efficiency, and underlying neuroprotective mechanisms.

Through comprehensive characterization involving in vitro release studies, rheological assessments, pharmacokinetic evaluations, and brain distribution imaging, we established that our delivery system exhibits sustained release characteristics and possesses significant capacity for nasal-to-brain transport. Additionally, we elucidated the mitochondrial-mediated antioxidant and anti-apoptotic pathways involved, offering new insights and a robust experimental foundation for optimizing neuroprotective formulations and applications of this compound. This study contributes to advancing the field of targeted drug delivery, particularly for neuroprotective agents, and underscores the potential of our PNIPAM-based system in enhancing therapeutic outcomes.

The determination of gelation temperature is a fundamental and crucial step in the preparation of thermosensitive hydrogels, as it directly influences whether the hydrogel can achieve the desired therapeutic efficacy under physiological conditions, such as nasal temperature [52]. LCST measurements indicated that the hydrogel successfully undergoes a sol-Gel phase transition between room temperature and nasal cavity temperature, thereby establishing a solid foundation for its potential application in nasal drug delivery systems. To further investigate the structural integrity and performance characteristics of taxifolin-loaded thermosensitive hydrogels, comprehensive physicochemical characterizations were carried out. SEM analysis revealed that the TF-GelGel possesses a uniform and well-defined pore structure, which reduces stress concentrations and enhances the overall mechanical properties. The regularity of the pores plays a key role in modulating the drug diffusion rate, thus facilitating controlled drug release. Furthermore, the smoothness of the pore walls contributes to minimizing physical damage to cells during the adhesion process, significantly improving the hydrogel biocompatibility.

FT-IR analysis confirmed the successful incorporation of taxifolin into the thermosensitive hydrogel matrix. Notably, this loading process did not induce any significant alterations to the core structure of the hydrogel or the characteristic functional groups of taxifolin. The spectrum clearly presents the characteristic absorption peak of the hydrogel polymer chain, along with distinct peaks corresponding to the aromatic ring C=C stretching vibration and the phenolic hydroxyl O-H stretching vibration of taxifolin. These observations suggest that the interaction between taxifolin and the hydrogel matrix is primarily driven by hydrogen bonding rather than chemical modification. Additional particle size distribution and thermogravimetric analysis further corroborate the uniformity and structural stability of the TF-Gel, supporting its potential for long-term, sustained drug release.

Rheological temperature scanning analysis revealed that TF-Gel undergoes a sol-Gel phase transition at 33 °C. At the physiological temperature of the nasal cavity (37 °C), the hydrogel exhibited excellent viscoelastic properties and structural stability. These characteristics, including its LCST and mechanical performance parameters, align well with the core requirements for nasal drug delivery systems, providing essential scientific and technical support for the development of efficient, stable nasal formulations. Specifically, temperature scanning experiments demonstrated that the structure of TF-Gel remains stable across various temperatures without signs of irreversible phase separation or degradation. Frequency scanning further confirmed that the hydrogel network possesses exceptional strength, capable of effectively resisting external stress. Shear rate tests indicated that the hydrogel maintained robust gel performance across a broad shear rate range, ensuring stable drug release in the nasal cavity. Additionally, stress–strain testing revealed that the deformation resistance of TF-Gel was significantly enhanced under various conditions. Strain–time tests at 25 °C showed that changes in modulus were primarily influenced by strain magnitude. At low strains, the elastic modulus dominated, contributing to the hydrogel’s superior shape recovery ability. However, at higher strains, while partial damage to the gel network occurred, leading to reduced elasticity, the hydrogel viscous dissipation mechanism remained active, indicating effective energy absorption. Furthermore, cycle stability tests confirmed that TF-Gel maintained stable rheological properties after five consecutive cycles, with no significant performance degradation, demonstrating the material’s excellent mechanical reversibility and fatigue resistance. Under constant sub-transition temperature conditions (25 °C), the thermosensitive behavior was not activated since the phase transition temperature threshold was not reached. As a result, modulus changes were independent of temperature fluctuations, further validating the threshold-dependent regulation of the hydrogel performance by temperature.

In order to further investigate the sustained-release behavior and biosafety of TF-Gel, in vitro nasal mucosal testing results showed a controlled and prolonged drug release profile, which can prolong the retention time of the drug in the nasal mucosa, reduce the frequency of administration, and improve patient compliance. Meanwhile, slow release can avoid excessive local drug concentration and reduce the risk of nasal mucosal irritation. The comprehensive evaluation results of biosafety showed that TF-Gel had good biocompatibility and hemocompatibility. The weight of mice increased steadily within 14 days without significant fluctuations, indicating that the hydrogel did not cause systemic toxicity or metabolic disorder. Histological examination of the nasal mucosa showed no cilia shedding and no inflammatory reaction, indicating that the hydrogel was not irritating to the nasal mucosa. There was no significant difference between the pathological sections of all organs and the control group, confirming the absence of distal organ toxicity and supporting the overall biological safety of TF-Gel.

To further investigate the brain-targeted delivery efficacy of TF-Gel, a series of pharmacokinetic studies was systematically conducted [53,54]. The experimental results demonstrate that the TF-Gel intranasal drug delivery system effectively overcomes the major challenges associated with traditional drug delivery methods for treating central nervous system (CNS) diseases, including poor BBB penetration and the requirement for frequent dosing [55,56]. TF-Gel significantly extends the circulation time of the drug in plasma and enhances its targeted delivery to brain tissue. This distinctive pharmacokinetic profile offers novel dosing strategies and therapeutic options for chronic neurological conditions, such as Alzheimer’s disease, which require sustained therapeutic drug concentrations.

Furthermore, TF-Gel exhibits exceptional sustained release properties, maintaining stable plasma drug concentrations for up to 24 h, while the drug remains in brain tissue for up to 48 h. Pharmacokinetic analysis revealed a significant prolongation in the T_1/2 of TF-Gel in the brain, reaching 22.96 h, and the MRT_0-t of the drug was extended to 17 h. These findings suggest that the dosing regimen could be optimized to a 3–5 day interval, significantly improving patient compliance and quality of life compared to the traditional daily dosing schedule. Additionally, a marked reduction in the Cmax was observed, while the area under the concentration–time curve (AUC_0-t) increased, indicating a substantial increase in the distribution of the drug to brain tissue. This shift in drug distribution reduces exposure to other tissues, thereby mitigating the potential risk of systemic side effects. Targeting efficiency parameters, including DTP and DTE, further confirmed the advantages of TF-Gel nasal administration for brain-targeted delivery. Collectively, these pharmacokinetic and bioavailability profiles support the strong clinical potential of TF-Gel as an effective delivery platform for chronic neurological disorders.

Given that taxifolin was mainly administered orally in previous studies [57], this study used small animal in vivo imaging technology to conduct an in-depth exploration of the brain targeting characteristics of the TF-Gel nasal delivery system. Through dynamic live fluorescence imaging of mice over a 48 h period, we compared the distribution of the drug following nasal versus oral administration. The results indicated that intranasal administration of TF-Gel led to rapid and preferential accumulation of the drug in brain tissue, as evidenced by strong fluorescence signals in the brain region. In contrast, after oral administration, the drug is initially distributed widely across peripheral organs, with a slower and significantly lower accumulation in the brain. This disparity can be attributed to the different pharmacokinetic pathways of the two administration routes. While oral administration requires the drug to traverse the circulatory system, exposing multiple organs before reaching the brain, nasal delivery bypasses the BBB directly through the olfactory and trigeminal nerve pathways, enabling targeted drug delivery to the brain. Notably, nasal administration also prolonged the retention time of the drug within the brain compared to the oral route. These findings provide compelling in vivo evidence for the superior brain targeting and delivery efficiency of the TF-Gel nasal delivery system, supporting its potential as an effective treatment strategy for chronic neurological diseases.

Numerous studies have indicated that the pathogenesis of Alzheimer’s disease is closely linked to mitochondrial dysfunction [58,59]. Under normal conditions, mitochondrial fusion and fission processes are in dynamic equilibrium, which plays a pivotal role in maintaining cellular energy metabolism and regulating apoptosis. When mitochondrial function is compromised, this balance is disrupted, leading to metabolic disorders and promoting apoptosis [60,61]. As the primary source of cellular energy, ATP is closely tied to mitochondrial function, and its levels reflect mitochondrial activity [62]. The current study revealed a significant decrease in ATP levels in the hippocampus of AD mice, suggesting severe impairment of mitochondrial oxidative phosphorylation. Treatment with TF-Gel significantly increased ATP content, indicating that TF-Gel effectively restores mitochondrial oxidative phosphorylation and enhances mitochondrial energy metabolism. Furthermore, the significant elevation in ROS and MDA levels in the AD group points to substantial oxidative stress damage. TF-Gel was able to reverse the abnormal levels of ROS and MDA, thereby demonstrating its potent antioxidant and mitochondria-protective effects.

Moreover, studies have shown that ATP levels are positively correlated with spatial memory ability, while ROS levels are inversely associated with the degree of cognitive impairment [63,64]. This relationship was confirmed by the results of the Morris water maze test, supporting the central role of mitochondrial dysfunction in the pathogenesis of AD. The TF-Gel group further validated its neuroprotective effects.

Mitochondrial fusion is regulated by the coordinated action of outer membrane fusion proteins Mfn1/Mfn2, together with the inner membrane fusion proteins Opa1, whereas mitochondrial fission is primarily mediated by Drp1 and Fis1 [65,66]. Western blot analysis revealed a marked reduction in the expression levels of Mfn1, Mfn2, and Opa1 in the AD group, alongside a significant increase in Drp1 and Fis1 expression, indicating substantial mitochondrial fragmentation. However, TF-Gel intervention significantly elevated the levels of fusion proteins and reduced the expression of fission proteins, suggesting that TF-Gel restores mitochondrial dynamic homeostasis and alleviates mitochondrial structural abnormalities.

Additionally, mitochondria play a central role in the intrinsic apoptosis pathway, with Cyto-C being a key factor in the initiation of apoptosis [67,68]. Western blot analysis demonstrated significantly elevated Cyto-C levels in the AD group, along with increased expression of apoptosis-related markers Bax, cleaved caspase-3, and cleaved caspase-9, while anti-apoptotic protein Bcl-2 levels were markedly decreased. After TF-Gel treatment, these alterations were notably reversed, suggesting that TF-Gel inhibits apoptosis through the mitochondrial pathway, thereby exerting its neuroprotective effects. Notably, the Gel group failed to elicit any beneficial effects on the measured parameters, showing no significant difference from the AD model group. This further supports that the therapeutic efficacy is specifically mediated by taxifolin.

The present study demonstrates the feasibility of a taxifolin-loaded thermosensitive hydrogel (TF-Gel) for intranasal administration and provides a foundational platform. However, it is crucial to recognize the challenges for clinical translation. While our findings are promising, achieving reliable functionality in more human-relevant models, engineering user-friendly delivery devices, and addressing the long-term stability of therapeutics within the hydrogel matrix are essential next steps. Additionally, a comprehensive understanding of in vivo degradation products and the measurement of hydrogel adhesive strength in vitro or in vivo are important future directions. Looking forward, the developed gel matrix can serve as a versatile platform for constructing more advanced hybrid delivery systems to address these limitations. A logical strategy would be to incorporate taxifolin into polymeric micelles designed for enhanced mucosal penetration, and then disperse them within this thermosensitive hydrogel. Such a hybrid approach could synergize the prolonged nasal residence of the Gel with the improved targeting capabilities of polymeric micelles, thereby addressing multiple biological barriers simultaneously for more efficient nose-to-brain delivery. Addressing these challenges and exploring hybrid systems will be the primary focus of our subsequent work to enhance the translational potential of our approach.

5. Conclusions

In this study, a novel taxifolin-based thermosensitive hydrogel delivery system was developed to achieve targeted brain delivery via the nasal–brain pathway, demonstrating significant efficacy in alleviating cognitive impairments associated with AD. Through a series of multidimensional characterization experiments, the physicochemical properties of the TF-Gel, including structural stability, particle size uniformity, and thermal stability, were comprehensively validated. These findings provide a robust scientific foundation for the potential of TF-Gel as an efficient brain-targeted drug delivery system. Further investigation into the three-dimensional mechanisms governing energy metabolism, mitochondrial dynamics, and cell apoptosis confirmed that TF-Gel can modulate mitochondrial function through multiple molecular targets. It effectively mitigates oxidative stress damage and neuronal apoptosis, which are key pathological processes in AD. The mechanism of action adheres to the principle of multi-pathway collaborative intervention, offering crucial experimental support for the development of AD treatment strategies focused on mitochondrial protection. But its deeper mechanism of action still needs to be further explored in subsequent research. Overall, these findings highlight the substantial potential of intranasal TF-Gel administration to overcome the limitations of conventional delivery systems and offer a promising framework for the development of brain-targeted treatments for neurodegenerative diseases such as Alzheimer’s disease.