Nasal injection type baicalein hydrogel as well as preparation method and application thereof

Abstract

The invention discloses nasal injection type baicalein hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving carboxymethyl chitosan in water, stirring to form a uniform carboxymethyl chitosan solution, then adding a 4-formylphenylboronic acid / baicalein solution, and then continuously stirring until yellow transparent hydrogel is formed, namely the nasal injection type baicalein hydrogel is obtained. The hydrogel has self-healing ability and proper viscosity, can be adhered to the epithelium of the nasal cavity and is continuously released in the microenvironment of the nasal cavity, so that the brain entering efficiency of the baicalein is remarkably improved; neurotoxic oligomers formed by abnormal aggregation of alpha-syn can be reduced; neuroinflammation can be relieved by regulating and controlling phenotypic polarization of microglial cells M2; oxidative stress can be improved by removing excessive ROS, dopaminergic neuron apoptosis is reduced, and the compound can be used for treating Parkinson's disease and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of drug delivery and relates to a nasal injection type baicalein hydrogel and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder. However, the gold-standard drug (levodopa) commonly used in clinical practice only alleviates motor dysfunction in PD patients, and currently lacks therapeutic strategies that can reverse PD progression. To overcome the growing societal burden of PD, the development of new anti-PD drugs is urgently needed. However, the development of new PD drugs faces two major challenges: the complex pathological mechanisms and the blood-brain barrier (BBB), which restricts drug access to the brain.

[0003] Existing evidence indicates that the progression of Parkinson's disease (PD) is closely associated with pathological changes such as oxidative stress, misfolding and aggregation of α-synuclein (α-syn), and neuroinflammation. Under pathological conditions of oxidative stress, neuronal mitochondrial function is impaired, leading to decreased cell metabolism and the inability of the energy-dependent ubiquitin protease system to clear intracellular toxic substances. Consequently, misfolded α-syn continuously assembles and aggregates within the cell body, ultimately forming neurotoxic α-syn oligomers that damage the nucleus, cell membrane, and lysosomes. Consequently, dopaminergic neurons undergo apoptosis under the influence of these pathogenic factors. The resulting cellular debris and prion-like amplification of α-syn oligomers can activate pattern recognition receptors in microglia. In a pathological microenvironment characterized by excessive reactive oxygen species (ROS), microglia become overactivated, producing a large number of inflammatory factors, causing neuroinflammation and further exacerbating disease progression. Under the influence of these multiple pathogenic factors, a large number of dopaminergic neurons are lost, thereby weakening nerve conduction to the striatum, leading to the dysfunction of dopamine metabolism, and ultimately causing PD patients to exhibit motor dysfunction. In addition, the spread of α-syn oligomers and neuroinflammation can also cause damage to other brain regions, causing PD patients to exhibit other disorders, such as reduced cognitive function. Therefore, blocking the development and mutual promotion of pathogenic factors such as oxidative stress, abnormal α-syn aggregation, and neuroinflammation is the key to reversing the progression of PD and is also an important research direction in the field of PD treatment.

[0004] The BBB is a physiological barrier that protects the central nervous system (CNS). While protecting the CNS, it also restricts drug access to the brain. Consequently, most therapeutic agents that demonstrate promising anti-PD therapeutic potential in vitro fail to replicate their efficacy in vivo. To overcome the BBB, a series of novel non-invasive brain drug delivery strategies have been developed in recent years, including reversible BBB opening through physical methods such as ultrasound and photothermal therapy, nanomedicine camouflage using biomembrane biomimetic technology, and intranasal injection. Intranasal drug delivery involves injecting drugs into the nasal cavity, allowing them to bypass the BBB through the olfactory epithelium / trigeminal nerve pathway and directly enter the CNS. Compared to other brain delivery strategies, intranasal injection has higher brain bioavailability and significantly reduces drug accumulation in other organs, potentially reducing the toxicity and increasing the efficacy of anti-PD therapeutics. To prolong drug retention in the nasal cavity and prevent rapid clearance by the nasal environment, drugs need to be formulated into injectable hydrogel systems with suitable mechanical strength, high viscosity, and good injectability, enabling them to adhere to the nasal mucosa and achieve sustained release into the brain. Summary of the Invention

[0005] In order to solve the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a method for preparing a nasal injection-type baicalein hydrogel.

[0006] Another object of the present invention is to provide a nasal injection-type baicalein hydrogel obtained by the above preparation method.

[0007] Another object of the present invention is to provide the use of the above-mentioned nasal injection-type baicalein hydrogel in the preparation of drugs for treating neurodegenerative diseases.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] A method for preparing a nasal injection-type baicalein hydrogel comprises dissolving carboxymethyl chitosan in water, stirring to form a uniform carboxymethyl chitosan solution, adding a 4-formylphenylboronic acid / baicalein solution, and then continuing to stir until a yellow transparent hydrogel is formed.

[0010] Preferably, the carboxymethyl chitosan is dissolved in water at a concentration of 1.5 to 2.5 w / v%.

[0011] More preferably, the carboxymethyl chitosan is dissolved in water at a concentration of 2 w / v%.

[0012] Preferably, the 4-formylphenylboronic acid is dissolved in water at a concentration of 0.5 to 0.7 w / v%.

[0013] More preferably, the 4-formylphenylboronic acid is dissolved in water at a concentration of 0.6 w / v%.

[0014] Preferably, the baicalein is dissolved in water at a concentration of 0.04 to 0.4 w / v%.

[0015] More preferably, the baicalein is dissolved in water at a concentration of 0.16 w / v%.

[0016] Preferably, the volume ratio of the carboxymethyl chitosan solution to the 4-formylphenylboronic acid / baicalein solution is 1-1.2:1-1.2.

[0017] Preferably, the volume ratio of the carboxymethyl chitosan solution to the 4-formylphenylboronic acid / baicalein solution is 1:1.

[0018] Preferably, the stirring speed is 250-350 rpm; more preferably 300 rpm.

[0019] A nasal injection type baicalein hydrogel is obtained by the above preparation method. The nasal injection type baicalein hydrogel has a pore size range of 90 to 150 μm and a phase transition temperature of 45 to 50°C.

[0020] Application of the nasal injection type baicalin hydrogel in the preparation of drugs for treating neurodegenerative diseases.

[0021] Furthermore, the neurodegenerative diseases include but are not limited to Parkinson's disease.

[0022] Furthermore, the medicine is a preparation for nasal administration.

[0023] The present invention has the following advantages and effects compared to the prior art:

[0024] The nasal injection-type baicalein hydrogel provided by the present invention has self-healing ability and appropriate viscosity, can adhere to the nasal epithelium, and is continuously released in the nasal microenvironment, significantly improving the efficiency of baicalein entering the brain; can reduce the neurotoxic oligomers formed by abnormal aggregation of α-syn; can alleviate neuroinflammation by regulating the polarization of the M2 phenotype of microglia; can improve oxidative stress by clearing excessive ROS and reduce dopaminergic neuron apoptosis, and can be used to treat Parkinson's disease, with good application prospects.

[0025] The materials used in the present invention are all approved by the FDA, have excellent biocompatibility and high safety.

[0026] The invention has a simple process and a fast preparation speed and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The results of baicalein inhibiting the phase separation of α-syn protein; (A) Computer simulation results of the interaction between baicalein and α-syn protein molecules; (B) Bright field imaging detection of the inhibitory effect of 0, 1, and 10 μM baicalein on the formation of α-syn phase separation droplets; (C) Coomassie Brilliant Blue detection of the inhibitory effect of 0, 1, and 10 μM baicalein on the formation of α-syn phase separation droplets.

[0028] Figure 2 The results of the characterization of the physicochemical properties of nasal injection-type baicalein hydrogel; (A) Injectability, self-healing and adhesion of nasal injection-type baicalein hydrogel; (B) Scanning electron microscopy imaging of nasal injection-type baicalein hydrogel; scale: 100μm; (C) Thermosensitive properties of nasal injection-type baicalein hydrogel.

[0029] Figure 3 The figures show the drug release characteristics of nasal injection-type baicalein hydrogel; (A) pH-responsive drug release characteristics of nasal injection-type baicalein hydrogel; (B) ROS-responsive drug release characteristics of nasal injection-type baicalein hydrogel.

[0030] Figure 4 Figure 2 is the in vitro efficacy validation result of nasal injection hydrogel; in the figure, BA refers to baicalein, and CAB2 refers to nasal injection baicalein hydrogel; (A) Nasal injection baicalein hydrogel inhibits the formation of α-syn oligomers and its nuclear translocation in PC12 cells; Neuroprotective effect of nasal injection baicalein hydrogel intervention on PC12 cells: (B) live / dead cell fluorescence imaging and (C) cell activity detection; (D) Regulation of energy metabolism of PC12 cells by nasal injection baicalein hydrogel; (E) Regulatory effect of nasal injection baicalein hydrogel on the relative expression levels of cytokines and their surface markers in BV2 cells.

[0031] Figure 5 This figure shows the results of verifying the brain drug delivery efficiency of nasal baicalein hydrogel at the animal level; in the figure, BA refers to baicalein, and CAB2 refers to nasal baicalein hydrogel; after nasal injection of baicalein solution and nasal baicalein hydrogel, the pharmacokinetic curves of (A) olfactory bulb, (B) hippocampus, (C) brain without hippocampus, and (D) cerebellum.

[0032] Figure 6 Figure 2 is the efficacy verification result of nasal injection type baicalin hydrogel at the animal level; in the figure, BA refers to baicalin, and CAB2 refers to nasal injection type baicalin hydrogel; (A) Effect of nasal injection type baicalin hydrogel intervention on the gait of PD mice; open field test detects the effect of nasal injection type baicalin hydrogel intervention on the motor function of PD mice: (B) mouse movement trajectory diagram; (C) total distance of mouse movement; (D) mouse movement speed.

[0033] Figure 7 The figures show the effects of nasal baicalein hydrogel intervention on the pathological environment of PD mice; in the figures, BA refers to baicalein, and CAB2 refers to nasal baicalein hydrogel; (A) Effects of nasal baicalein hydrogel intervention on the number of dopaminergic neurons in the substantia nigra of PD mice; (B) Effects of nasal baicalein hydrogel intervention on the expression levels of TH and α-syn proteins in the midbrain of PD mice; (C) Effects of nasal baicalein hydrogel intervention on the expression levels of cytokines and M2 microglial cell surface markers in the midbrain of PD mice. DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0035] Example 1: Inhibitory effect of baicalein on liquid-liquid phase separation of α-synuclein (α-syn)

[0036] (1) Docking of baicalin and α-syn molecules

[0037] Protein preparation: α-syn protein, the 3D structure with the PDB database number 7OZG (resolution 2.5A) was selected for molecular docking, and AutoDock Tools and Pymol were used to perform operations such as dehydration, removal of original ligand small molecules, hydrogenation, and charge distribution. Small molecule preparation: The 3D structure of baicalein (Baicalein, PubChem CID: 5281605) was obtained from the Pubchem website and pre-processed with AutoDock Tools such as energy minimization. Molecular docking: The default docking parameters exhaustiveness = 24, num_modes = 9 were set, the docking pocket coordinates were the entire protein range, and autodock vina was used for molecular docking. Visualization analysis: The conformation with the most stable binding energy of the molecular docking results was exported, and the action site and force of the protein-small molecule complex were analyzed using the PLIP database, and visualized using Pymol. The results are as follows Figure 1 As shown in A, there are four binding sites between baicalein and the target protein α-syn.

[0038] (2) Baicalein inhibits the formation of large α-syn droplets

[0039] Liquid-liquid phase separation buffer was prepared by mixing 50mM MES buffer and 50mM Tris to a pH of 5.5. PEG8000 was added to prepare a 15% PEG8000 MES-Tris buffer. Baicalein solutions at 0, 1, and 10μM were prepared in this buffer, followed by the addition of purified α-syn protein to a concentration of 8μM. The resulting solutions were added to a 96-well plate, with 150μL of protein per well. The formation of large droplets was observed daily under an optical microscope for 4 days. Figure 1 As shown in Figure B, the buffer system without baicalein can form a significant phase-separated large liquid, while the presence of baicalein significantly interferes with the formation of large liquids, and the effect of higher concentrations of baicalein on inhibiting phase-separated large droplets is more significant. The phase-separated large droplets formed in each group were collected, and loading buffer was added and protein denaturation was performed. Then, gel electrophoresis was performed, and the aggregation phenomenon of α-syn was suggested by Coomassie Brilliant Blue staining test. The results are shown in Figure 2. Figure 1 As shown in Figure C, in the group without baicalein, the Coomassie blue stain of α-syn was the darkest, and a clear "tailing" phenomenon was observed, indicating the presence of α-syn oligomers, while this phenomenon was not observed in the group containing baicalein. These results demonstrate that baicalein can effectively inhibit the aggregation of α-syn.

[0040] Example 2: Preparation and characterization of nasal injection-type baicalein hydrogel

[0041] (1) Preparation of nasal injection-type baicalein hydrogel

[0042] Carboxymethyl chitosan was dissolved in ultrapure water at a concentration of 2 w / v%. The solution was stirred at 300 rpm at room temperature to form a homogeneous solution. Equal volumes of 4-formylphenylboronic acid and baicalein solutions were then added. The concentrations of 4-formylphenylboronic acid and baicalein were 0.6 w / v% and 0.16 w / v%, respectively. A yellow, transparent hydrogel was formed by stirring at 300 rpm at room temperature. The resulting gel contained carboxymethyl chitosan at a concentration of 1 w / v%, 4-formylphenylboronic acid at a concentration of 0.3 w / v%, and baicalein at a concentration of 0.08 w / v%.

[0043] Injectability Test: The nasal baicalin hydrogel was injected into a 1mL syringe. The hydrogel was observed to be able to be smoothly ejected from the needle end of the syringe when pushed by the syringe plunger, and a camera was used to record the results. Self-Healing Test: The nasal baicalin hydrogel was cut into two parts. The cut surfaces were then placed together and the hydrogel was observed to heal into one piece at room temperature. Adhesion Test: The nasal baicalin hydrogel was adhered to a gloved finger. The adhesion was observed by bending and turning the finger, and a camera was used to record the results.

[0044] like Figure 2As shown in A, the obtained nasal injection-type baicalein hydrogel has good injectability, self-healing ability and adhesion, meeting the requirements of nasal injection.

[0045] (2) Characterization of nasal injection-type baicalein hydrogel

[0046] The nasal injection-type baicalein hydrogel was placed in a -20°C refrigerator and frozen overnight, then treated in a freeze dryer for 12 hours. It was then taken out and the top layer was cut off with a blade. It was then attached to the scanning electron microscope sample stage with conductive glue. After gold spraying, the morphology was observed using a scanning electron microscope.

[0047] The results are as follows Figure 2 As shown in Figure B, the nasal injection-type baicalin hydrogel exhibits a significant porous network structure with a pore size of 145.4±44.75μm.

[0048] The nasal baicalein hydrogel was placed on the sample plate of the rheometer. The temperature range was set to 25-60°C, the strain was 1%, and the angular frequency was 6.28 rad / s. The storage modulus (G') and loss modulus (G") of the nasal baicalein hydrogel were tested with temperature.

[0049] The results are as follows Figure 2 As shown in Figure C, when the temperature exceeds 48.6°C, the loss modulus of the nasal baicalein hydrogel is greater than the storage modulus, indicating that 48.6°C is the phase transition temperature of the nasal baicalein hydrogel. When the temperature is below 48.6°C, the nasal baicalein hydrogel can remain in a gel state, while at temperatures above 48.6°C, the nasal baicalein hydrogel transforms into a liquid state. Therefore, the nasal baicalein hydrogel can maintain a stable gel structure at physiological temperature, meeting the requirements of nasal injection.

[0050] 1 mL of hydrogel was placed in a 5 mL centrifuge tube, and 3 mL of PBS solution (0.01 M) with pH values ​​of 7.4, 6.8, and 5.5 was added. The tube was shaken at 37°C and 100 rpm. After a predetermined time, 300 μL of the release solution was removed and 300 μL of fresh PBS solution was added to the release system to maintain a constant volume. The H2O2-responsive drug release test used PBS buffer with a pH of 6.8 containing 1 mM H2O2 as the release medium. The other steps were the same as those for the pH-responsive drug release process. Finally, the drug release amount of the nasal injection-type baicalein hydrogel was analyzed using a UV-visible spectrophotometer.

[0051] The results are as follows Figure 3 As shown in the results, the nasal injection type baicalein hydrogel has a faster drug release rate in an acidic and ROS-containing environment.

[0052] Comparative Examples 1 to 3:

[0053] The procedure was the same as in Example 2, except that in step 1, in the preparation of the nasal-injectable baicalein hydrogel, the baicalein concentrations in Comparative Examples 1-3 were 0.1 w / v%, 0.2 w / v%, and 0.6 w / v%, respectively. The results showed that no baicalein hydrogel could be formed at a baicalein concentration of 0.1 w / v%. When the baicalein concentrations were 0.2 w / v% and 0.6 w / v%, the baicalein hydrogels formed were too strong and unsuitable for injection.

[0054] Compared with Comparative Examples 1 to 3, the concentration of baicalein added in Example 2 is 0.16 w / v%, which can form a stable baicalein hydrogel and has good injectability, suitable for nasal injection. The present invention finally selects the addition amount of baicalein in the nasal injection type baicalein hydrogel to be 0.16 w / v%.

[0055] Comparative Examples 4 to 6:

[0056] The procedure was the same as in Example 2, except that in step 1, in the preparation of the nasal-injectable baicalein hydrogel, the carboxymethyl chitosan concentrations in Comparative Examples 4-6 were 1 w / v%, 1.5 w / v%, and 4 w / v%, respectively. Results showed that baicalein hydrogels could not form when the baicalein concentrations were 1 w / v% and 1.5 w / v%. While baicalein hydrogels formed rapidly when the concentration was 4 w / v%, the resulting gels were too strong and not injectable.

[0057] Compared with Comparative Examples 4 to 6, the carboxymethyl chitosan added at a concentration of 2 w / v% in Example 2 can form a stable baicalein hydrogel with good injectability and is suitable for nasal injection. The present invention ultimately selects 2 w / v% carboxymethyl chitosan as the raw material to prepare the nasal injection type baicalein hydrogel.

[0058] Comparative Examples 7 to 9

[0059] The procedure was the same as in Example 2, except that in step 1, in preparing the nasal injection-type baicalein hydrogel, the concentrations of 4-formylphenylboronic acid added in Comparative Examples 7 to 9 were 0.2 w / v%, 0.4 w / v%, and 1 w / v%, respectively. The results showed that when the 4-formylphenylboronic acid concentration was 2 w / v%, no baicalein hydrogel could be formed; when the 4-formylphenylboronic acid concentration was 4 w / v%, no stable baicalein hydrogel could be formed; and when the 4-formylphenylboronic acid concentration was 10 w / v%, a baicalein hydrogel could be rapidly formed, but undissolved 4-formylphenylboronic acid crystals were present in the formed hydrogel.

[0060] Compared with Comparative Examples 7 to 9, Example 2, in which 4-formylphenylboronic acid was added at a concentration of 0.6 w / v%, was able to form a stable baicalein hydrogel. The hydrogel was a yellow, transparent colloid, free of undissolved 4-formylphenylboronic acid crystals, and had good injectability, making it suitable for nasal injection. The present invention ultimately selected 4-formylphenylboronic acid at a concentration of 0.6 w / v% to prepare a nasal injectable baicalein hydrogel.

[0061] Example 3: Validation of the efficacy of nasal injection-type baicalein hydrogel at the cellular level

[0062] (1) Nasal injection of baicalin hydrogel inhibits α-syn protein phase separation at the cellular level

[0063] Rat adrenal medullary pheochromoma cells PC12 were seeded in 12-well plates and the intervention was started after the cells grew to an appropriate density. PC12 cells were first pretreated with 12.5 μg / mL baicalein or nasal baicalein hydrogel for 4 hours, and then the culture medium was replaced with 1 mM MPP. + with 10 μM Cu 2+ The cells were then incubated for 36 hours in a drug-containing medium (the concentrations of baicalein and nasal baicalein hydrogel were the same as those used for pretreatment). The PC12 cells in the well plates were then fixed, stained with α-syn immunofluorescence and DAPI, and finally imaged using a confocal fluorescence microscope.

[0064] The results are as follows Figure 4 As shown in A, in MPP + +Cu 2+ In the nasal injection group, α-syn formed more large droplets, indicating that the number of α-syn oligomers increased and nuclear transfer occurred significantly, while baicalein and nasal injection baicalein hydrogel significantly inhibited the formation of α-syn oligomers and their nuclear transfer, indicating that baicalein and nasal injection baicalein hydrogel can effectively inhibit the liquid-liquid phase separation of α-syn, thereby reducing the cytotoxicity caused by α-syn neurotoxic oligomers.

[0065] (2) Neuroprotective effect of nasal baicalein hydrogel at the cellular level

[0066] PC12 cells were seeded in confocal microplates and intervention was started after the cells grew to an appropriate density. PC12 cells were first pretreated with 12.5 μg / mL baicalein or nasal baicalein hydrogel for 4 hours, and then the culture medium was replaced with 2 mM MPP. + The cells were then incubated for another 24 hours in a drug-containing culture medium (the concentrations of baicalein and nasal baicalein hydrogel were the same as those used for pretreatment). The PC12 cells in the well plates were then stained for live and dead cells and imaged using a confocal fluorescence microscope.

[0067] The results are as follows Figure 4 As shown in B, MPP + Most cells in the nasal injection group died, while baicalein and baicalein hydrogel for nasal injection effectively increased the cell survival rate.

[0068] PC12 cells were seeded in 96-well plates and the intervention was started after the cells grew to an appropriate density. PC12 cells were first pretreated with gradient concentrations of baicalein (50, 25, 12.5, 6.25, 3.125 μg / mL) or nasal baicalein hydrogel for 4 hours, and then the culture medium was replaced with 2 mM MPP. + The cells were incubated for another 24 hours in a drug-containing medium (the concentrations of baicalein and nasal baicalein hydrogel were the same as those used in the pretreatment). The cell viability of PC12 cells was then detected using a CCK-8 kit.

[0069] The results are as follows Figure 4 As shown in middle C, baicalein and nasal baicalein hydrogel can effectively improve the cell activity of PC12, and the efficacy of nasal baicalein hydrogel is better than that of baicalein.

[0070] PC12 cells were seeded in 96-well plates at a density of 18,000 cells / well. After 24 hours of cell attachment, they were treated with 12.5 μg / mL baicalein and nasal baicalein hydrogel at 37°C for 4 hours. The culture medium was then replaced with 2 mM MPP. + Cells were pretreated for 2 hours with drug-containing culture medium (the concentrations of baicalein and nasal baicalein hydrogel were the same as those used for pretreatment). Before the mitochondrial stress test, the cell culture medium in all experimental groups was replaced with 175 μL of FX medium (Agilent Technologies FX Assay-specific modified DMEM containing 5.5 mM glucose, 1 mM pyruvate, and 2 mM glutamine). After one buffer wash and a culture medium change, cells in each well were equilibrated in a 37°C non-CO2 incubator for 1 hour. The 96-well plates were then subjected to a mitochondrial stress test to analyze mitochondrial energy metabolism and cellular metabolic profiles. This test was performed by sequential injection of the following mitochondrial inhibitors: oligomycin A (1 μM), an ATP synthase inhibitor; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM), an uncoupler; and a mixture of antimycin A (1 μM) and rotenone (1 μM), which inhibits complexes I and III of the mitochondrial respiratory chain to block mitochondrial oxygen consumption.

[0071] The results are as follows Figure 4 As shown in D, MPP + It significantly inhibited the mitochondrial metabolism level of PC12 cells, while baicalein and nasal injection baicalein hydrogel could significantly improve the mitochondrial metabolism level of PC12 cells, and the effect of nasal injection baicalein hydrogel was better than baicalein.

[0072] (3) Anti-neuroinflammatory effect of nasal baicalein hydrogel at the cellular level

[0073] Mouse microglial BV2 cells were seeded in 12-well plates and intervention was initiated after the cells grew to an appropriate density. PC12 cells were first pretreated with 12.5 μg / mL baicalein or intranasal baicalein hydrogel for 4 hours. The culture medium was then replaced with 100 ng / mL lipopolysaccharide-containing medium (the concentrations of baicalein and intranasal baicalein hydrogel were the same as those used for pretreatment) and incubated again for 24 hours. RNA from BV2 cells in each well was then extracted using Trizol reagent, and the transcript levels of IL-1β, TNF-α, Arg-1, YM, CD206, IL-4, IL-10, and TGF-β were measured.

[0074] The results are as follows Figure 4 As shown in Figure E, compared with the control group, the transcription levels of proinflammatory factors in the LPS group were significantly increased, while the transcription levels of microglial M2 phenotype markers and anti-inflammatory factors were significantly decreased. Conversely, under the intervention of baicalein and nasal baicalein hydrogel, the transcription levels of BV2 cell M2 phenotype markers and anti-inflammatory factors were significantly increased, while the transcription levels of proinflammatory factors were significantly improved. This indicates that baicalein and nasal baicalein hydrogel can improve LPS-induced neuroinflammation at the cellular level in vitro.

[0075] Example 4: Brain drug delivery efficiency of nasal injection-type baicalein hydrogel at the animal level

[0076] C57BL / 6 mice were intranasally injected with 10 μL of baicalein and a baicalein hydrogel (0.6 mg / kg). The olfactory bulb, cerebrum, and cerebellum were harvested at 0.25, 1, 2, 4, 6, 8, 12, and 24 hours after administration, and the hippocampus was isolated from the brain. Each tissue was then homogenized and centrifuged at 13,000 × g for 10 minutes. The supernatant was then collected and analyzed for baicalein content by LC-MS.

[0077] The results are as follows Figure 5 As shown in the results, compared with nasal injection of free baicalein solution, nasal injection of baicalein hydrogel can significantly increase the drug content in the olfactory bulb, brain and cerebellum.

[0078] Example 5: Validation of the efficacy of nasal injection-type baicalein hydrogel at the animal level

[0079] (1) Construction of PD mouse model and drug administration regimen

[0080] C57BL / 6 male mice were randomly divided into five groups: 1) Control, 2) MPTP, 3) L-DOPA, 4) BA, and 5) CAB2. MPTP·HCl dissolved in 0.9% sodium chloride injection was intraperitoneally injected for 5 consecutive days to establish a PD model. The L-DOPA group received a daily intraperitoneal injection of 18 mg / kg of L-DOPA. The BA and CAB2 groups received a daily intranasal injection of the corresponding drug at a dose of 0.6 mg / kg, 10 μL per dose. After administration, gait analysis and open field test were performed on mice in each group. Frozen sections of midbrain tissue were collected and immunofluorescence staining of tyrosine hydroxylase (TH)-positive neurons, a marker of DA neurons, was performed. The expression levels of TH and α-syn in the midbrain were detected by Western blotting. In addition, the transcription levels of CD86, TNF-α, IL-1β, IL-6, CD206, IL-4, IL-10, and TGF-β were detected by RT-PCR.

[0081] (2) Gait analysis and behavioral tests of mice in each group

[0082] like Figure 6 As shown, gait analysis of each group of mice showed that the MPTP group had a significantly disordered gait, while L-DOPA, BA, and CAB2 intervention helped improve the mice's gait disorder. In the open field test, the MPTP group mice showed the least movement distance and movement speed, indicating that the mice's motor function was impaired. Similarly, L-DOPA, BA, and CAB2 intervention also showed improved motor function. Among them, the motor function of the CAB2 group mice was similar to that of the L-DOPA group. These results show that CAB2 can restore the motor dysfunction of PD mice.

[0083] (3) Pathological analysis of the brains of mice in each group

[0084] like Figure 7As shown, further frozen section results showed that the number of dopaminergic neurons in MPTP-treated mice was reduced, while the number of dopaminergic neurons in the L-DOPA, BA, and CAB2 groups was larger, and the number of dopaminergic neurons in the CAB2 group was similar to that in the control group, indicating that CAB2 can reduce the neurotoxic effect of MPTP on dopaminergic neurons. In addition, WB detection results once again proved that CAB2 can improve the MPTP-induced decrease in TH levels in the mouse midbrain. At the same time, CAB2 also showed a good α-syn inhibitory effect at the in vivo level, which is consistent with the results of in vitro studies. RT-PCR results also showed that CAB2 can significantly improve the MPTP-induced microglial M1 phenotype differentiation and pro-inflammatory cytokine expression. Conversely, CAB2 can promote microglial M2 phenotype differentiation and the secretion of anti-inflammatory factors, thereby improving neuroinflammation in the PD brain.

[0085] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a nasal injection-type baicalein hydrogel, characterized by: Carboxymethyl chitosan was dissolved in water and stirred to form a uniform carboxymethyl chitosan solution, followed by the addition of 4-formylphenylboronic acid / baicalein solution. The solution was then stirred until a yellow transparent hydrogel was formed.

2. The method for preparing the nasal injection-type baicalein hydrogel according to claim 1, wherein: The carboxymethyl chitosan is dissolved in water at a concentration of 1.5-2.5 w / v%.

3. The method for preparing the nasal injection-type baicalein hydrogel according to claim 1, wherein: The 4-formylphenylboronic acid is dissolved in water at a concentration of 0.5 to 0.7 w / v%; The baicalein is dissolved in water at a concentration of 0.04-0.4w / v%.

4. The method for preparing the nasal injection-type baicalein hydrogel according to claim 1, wherein: The volume ratio of the carboxymethyl chitosan solution to the 4-formylphenylboronic acid / baicalein solution is 1-1.2:1-1.

2.

5. The method for preparing the nasal injection-type baicalein hydrogel according to any one of claims 1 to 4, characterized in that: The carboxymethyl chitosan is dissolved in water at a concentration of 2 w / v%; The 4-formylphenylboronic acid is dissolved in water at a concentration of 0.6 w / v%; The baicalein is dissolved in water at a concentration of 0.16 w / v%; The volume ratio of the carboxymethyl chitosan solution to the 4-formylphenylboronic acid / baicalein solution is 1:

1.

6. The method for preparing the nasal injection-type baicalein hydrogel according to any one of claims 1 to 4, characterized in that: The stirring speed is 250-350 rpm.

7. A nasal injection-type baicalein hydrogel, characterized by: The method is obtained by the preparation method described in any one of claims 1 to 6.

8. Use of the nasal injection type baicalein hydrogel according to claim 7 in the preparation of drugs for treating neurodegenerative diseases.

9. The use according to claim 8, characterized in that: The neurodegenerative diseases include Parkinson's disease.

10. The use according to claim 8 or 9, characterized in that: The medicine is a nasal administration preparation.

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