A hydrogel and its preparation method and application

Abstract

The present invention relates to a hydrogel, a preparation method thereof and an application thereof. A triblock polymer (F127-CHO) of aldehyde group-terminated poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) forms a dynamically covalently crosslinked polymer network with carboxymethyl chitosan (CMCS), and a black phosphorus-methylene blue nanocomposite is in-situ loaded to prepare the hydrogel. The hydrogel has temperature responsiveness, self-healing and injectable functions, forms an in-situ gel after being injected into the nasal cavity, avoids being rapidly cleared by the nasal mucosa, slowly and controllably releases drugs, and nasal administration can bypass the blood-brain barrier and directly deliver the drugs into the brain, significantly improving the bioavailability of the brain tissue. The black phosphorus nanosheets can not only load the drug methylene blue, but also capture oxygen free radicals, playing a neuroprotective role in antioxidative stress. The present invention provides an effective nasal administration preparation for the treatment of brain diseases, and has good clinical application prospects and development value. 106 - poly(propylene glycol) 70 - poly(ethylene glycol) 106 The triblock polymer (F127-CHO) of aldehyde group-terminated poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) forms a dynamically covalently crosslinked polymer network with carboxymethyl chitosan (CMCS), and a black phosphorus-methylene blue nanocomposite is in-situ loaded to prepare the hydrogel. The hydrogel has temperature responsiveness, self-healing and injectable functions, forms an in-situ gel after being injected into the nasal cavity, avoids being rapidly cleared by the nasal mucosa, slowly and controllably releases drugs, and nasal administration can bypass the blood-brain barrier and directly deliver the drugs into the brain, significantly improving the bioavailability of the brain tissue. The black phosphorus nanosheets can not only load the drug methylene blue, but also capture oxygen free radicals, playing a neuroprotective role in antioxidative stress. The present invention provides an effective nasal administration preparation for the treatment of brain diseases, and has good clinical application prospects and development value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical nanomaterials, and relates to a hydrogel and its preparation method and application. Background Art

[0002] Neurodegenerative diseases occur frequently in the middle-aged and elderly, manifested as neuronal loss and myelin degeneration, ultimately leading to abnormal nervous system function. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), Amyotrophic lateral sclerosis (ALS), Huntington disease (HD), etc. Among them, Alzheimer's disease is the most common neurodegenerative disease, and its pathological features are extracellular amyloid protein deposition, neurofibrillary tangles formed by hyperphosphorylated Tau protein in cells, neuron cell death, and microglial hyperplasia, etc. However, the pathogenesis of AD is complex, and there is still no effective cure method so far. Currently, multiple clinical trials targeting Aβ cannot delay or prevent disease progression, and more and more researchers have shifted their research focus to other therapeutic targets related to AD, such as targeting Tau protein, anti-inflammation, reducing oxidative stress, improving metal ion metabolic disorders, etc.

[0003] The existence of the Blood Brain Barrier (BBB) has always been a problem in brain drug delivery. It strictly controls the substance exchange between the brain and the blood, and at the same time also hinders drugs from entering the brain to play an effective therapeutic role. Intranasal administration is a non-invasive and safe method that can bypass the blood brain barrier and directly enter the brain through the olfactory and trigeminal nerve pathways to achieve brain-targeted drug delivery. However, some drugs have poor membrane penetration ability, and are easily degraded by enzymes in the nasal cavity and cleared by nasal cilia after being administered in solution form, reducing the administration efficiency. Methylene blue can inhibit the aggregation of Tau protein and decompose Tau protofibrils, and has a therapeutic effect on Alzheimer's disease. However, it diffuses rapidly after entering the brain and cannot effectively stay in the brain to play a therapeutic role, with low bioavailability. Therefore, it is necessary to develop a nasal administration preparation that can prolong the contact time between the drug and the nasal mucosa, reduce drug clearance, and improve the nasal absorption and brain-targeted delivery efficiency of the drug. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a hydrogel with temperature responsiveness, self-healing and injectable functions, which can form an in-situ gel after being injected into the nasal cavity, avoid being rapidly cleared by the nasal mucosa, slowly control the release of drugs, and can bypass the blood-brain barrier to directly deliver the drugs into the brain through nasal administration, significantly improving the bioavailability of the brain tissue, as well as a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a preparation method of a hydrogel, including the following steps:

[0007] (1) Adding the aldehyde group-terminated poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock polymer F127-CHO to the dispersion liquid A of the black phosphorus nanocomposite loaded with methylene blue, dissolving to obtain a mixture A; 106 -poly(propylene glycol) 70 -poly(ethylene glycol) 106 Adding the aldehyde group-terminated poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock polymer F127-CHO to the dispersion liquid A of the black phosphorus nanocomposite loaded with methylene blue, dissolving to obtain a mixture A;

[0008] (2) Adding carboxymethyl chitosan to the dispersion liquid B of the black phosphorus nanocomposite loaded with methylene blue, dissolving to obtain a mixture B;

[0009] (3) Mixing the mixture A and the mixture B evenly, and obtaining the hydrogel after sufficient reaction;

[0010] The concentration of the black phosphorus nanocomposite loaded with methylene blue in the dispersion liquid A of the black phosphorus nanocomposite loaded with methylene blue is the same as that in the dispersion liquid B of the black phosphorus nanocomposite loaded with methylene blue;

[0011] The mass ratio of the F127-CHO to the carboxymethyl chitosan is (1-4):4.

[0012] In the step (3), the conditions for mixing include but are not limited to slow stirring in an ice-water bath, and the reaction condition is reaction at 37°C for 12 h.

[0013] A hydrogel is an insoluble polymer with a three-dimensional hydrophilic network crosslinked by water-soluble polymers. It has a good three-dimensional structure and a structure similar to the extracellular matrix, with good biocompatibility, and is widely used in technical fields such as wound dressings, drug sustained-release matrices, and human soft tissue filling. A nasal administration gel preparation with an appropriate phase transition temperature can rapidly form a gel after contacting the nasal mucosa, can serve as a drug reservoir, increase the residence time of the drug in the nasal cavity, and minimize mucociliary clearance, slowly control the release of drugs, and further improve the nasal absorption efficiency of the drug and the bioavailability of the brain tissue.

[0014] Black phosphorus (BP) is a new star in the family of two-dimensional materials. Its unique wrinkled structure endows it with a larger specific surface area, which is beneficial for drug loading. In addition, BP has great reactivity towards reactive oxygen species, can scavenge various types of oxygen free radicals both in vivo and in vitro, and is ultimately degraded into non-toxic phosphite ions and phosphates, showing excellent biocompatibility and having good application prospects in the treatment of neurodegenerative diseases.

[0015] Methylene blue (MB) can inhibit the aggregation of Tau protein and decompose Tau fibrils, showing a therapeutic effect on Alzheimer's disease. However, after entering the brain, it diffuses rapidly and cannot effectively stay in the brain to play a therapeutic role, resulting in low bioavailability. Methylene blue binds to black phosphorus through electrostatic adsorption and hydrophobic interaction to form a black phosphorus-methylene blue nanocomposite, avoiding rapid drug release. At the same time, black phosphorus nanosheets (BP NSs) have a biological effect of scavenging reactive oxygen species and can synergistically play a neuroprotective role with methylene blue.

[0016] The production process of the preparation method provided by the present invention is relatively simple. The solvent involved in synthesizing the composite hydrogel is water, which is non-toxic and environmentally friendly, enabling low-cost and large-scale production.

[0017] As a preferred embodiment of the preparation method of the hydrogel of the present invention, the mass ratio of F127-CHO to carboxymethyl chitosan is 3:4.

[0018] The hydrogel prepared when the mass ratio of F127-CHO to carboxymethyl chitosan is 3:4 has good temperature responsiveness, shear-thinning characteristics, can be used for injection administration, and has good self-healing ability.

[0019] As a preferred embodiment of the preparation method of the hydrogel of the present invention, in the dispersion A of the black phosphorus nanocomposite loaded with methylene blue and the dispersion B of the black phosphorus nanocomposite loaded with methylene blue, the concentration of the black phosphorus nanocomposite loaded with methylene blue is 0.1 - 3 mg / mL.

[0020] The volumes of the dispersion A of the black phosphorus nanocomposite loaded with methylene blue and the dispersion B of the black phosphorus nanocomposite loaded with methylene blue are 0.5 mL.

[0021] As a preferred embodiment of the preparation method of the hydrogel of the present invention, the preparation method of the black phosphorus nanocomposite loaded with methylene blue includes the following steps:

[0022] S1. Add an aqueous solution of methylene blue to the aqueous dispersion of black phosphorus nanosheets and react under stirring in the dark to obtain a reaction product;

[0023] S2. Centrifuge the obtained reaction product, wash it with an aqueous solution, and then resuspend it in water to obtain a dispersion of black phosphorus nanocomposite loaded with methylene blue.

[0024] In the step S1, the concentration of black phosphorus nanosheets in the aqueous dispersion of black phosphorus nanosheets is 0.5 - 2 mg / mL, and the concentration of methylene blue in the aqueous solution of methylene blue is 1 - 3 mg / mL; the volume ratio of the aqueous dispersion of black phosphorus nanosheets to the aqueous solution of methylene blue is 1:(1 - 3).

[0025] In the step S1, the preferred concentration of black phosphorus nanosheets in the aqueous dispersion of black phosphorus nanosheets is 1 mg / mL, the preferred concentration of the aqueous solution of methylene blue is 2 mg / mL, the preferred volume ratio of the aqueous dispersion of black phosphorus nanosheets to the aqueous solution of methylene blue is 1:1.5, the solvent water is deoxygenated water, and the stirring condition is stirring at room temperature for 10 - 12 h.

[0026] As a preferred embodiment of the preparation method of the hydrogel of the present invention, the particle size of the black phosphorus nanosheets is 100 - 200 nm, and the thickness is 5 - 8 nm.

[0027] The preparation method of the black phosphorus nanosheets includes the following steps:

[0028] Disperse black phosphorus powder in an N-methylpyrrolidone solution (NMP) of supersaturated NaOH and sonicate, centrifuge to collect the supernatant to obtain black phosphorus nanosheets.

[0029] In the preparation method of the black phosphorus nanosheets, the dosage ratio of black phosphorus powder to NMP is 0.75 - 1 mg:1 mL, the sonication condition is probe sonication at 600 w under an ice bath condition of 4 - 10 °C for 10 - 12 h, after centrifugation, take the supernatant and sonicate it under the same condition for 10 - 12 h, centrifuge again, and the obtained precipitate is black phosphorus nanosheets (BP NSs). All the above operations are carried out in the dark.

[0030] As a preferred embodiment of the preparation method of the hydrogel of the present invention, the preparation method of F127-CHO includes the following steps:

[0031] S1. Weigh Pluronic and dissolve it in an organic solvent, add Dess-Martin oxidant, and stir and heat for an oxidation reaction.

[0032] S2. Take the filtrate obtained from the oxidation reaction in step S1, concentrate it to a viscous state, then add it to ice-cold diethyl ether and stir to produce a precipitate, cool it, centrifuge to collect the precipitate to obtain F127-CHO.

[0033] In step S1, the organic solvent includes but is not limited to dichloromethane, and the dissolution conditions include but are not limited to overnight dissolution. The reaction conditions of F127 and Dess-Martin are heating at 40°C for 24 h. In step S2, the volume of ice-cold diethyl ether is 100-150 mL, and the cooling methods include but are not limited to cooling in an ice-water bath for 1 h. After centrifuging to collect the precipitate, the final product is obtained by drying. The drying methods include but are not limited to vacuum drying at 40°C for 12 h.

[0034] Pluronic (F127) is an amphiphilic triblock copolymer that self-assembles into micelles in water and can be used as a dynamic cross-linking agent. Aldehyde-terminated F127 can undergo an aldehyde-amine condensation reaction with carboxymethyl chitosan to form dynamic reversible Schiff base bonds, endowing the gel with good mechanical properties, excellent self-healing properties, and injectability, making it suitable as a nasal drug delivery gel preparation. F127-CHO is obtained by oxidizing the hydroxyl group at the end of Pluronic with Dess-Martin reagent.

[0035] In a second aspect, the present invention provides a hydrogel prepared by the method for preparing the hydrogel described above.

[0036] Under the preparation conditions of the present invention, the obtained hydrogel has the characteristics of temperature sensitivity, injectability, and self-healing. After administration, it forms a gel that adheres to the nasal mucosa and serves as a drug reservoir, prolonging the residence time of the drug in the nasal mucosa and enabling the drug to be continuously absorbed into the brain. It can be used as an excellent gel preparation for nasal drug delivery.

[0037] In a third aspect, the present invention provides the application of the hydrogel in the preparation of drugs for treating neurodegenerative diseases.

[0038] As a preferred embodiment of the application of the hydrogel of the present invention in the preparation of drugs for treating neurodegenerative diseases, the neurodegenerative disease is Alzheimer's disease.

[0039] The hydrogel loaded with black phosphorus-methylene blue nanocomposites prepared by the present invention is expected to be used as a drug delivery strategy in the multi-directional and multi-dimensional nano biomedical field, such as diseases mainly characterized by brain lesions in Alzheimer's disease, Parkinson's disease, stroke, etc.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. The hydrogel loaded with black phosphorus-methylene blue nanocomposites prepared by the present invention loads black phosphorus-methylene blue nanosheets into the three-dimensional network structure of the gel. The prepared composite hydrogel has dynamic chemical bonds and has the characteristics of temperature sensitivity, injectability, and self-healing. After administration, it forms a gel that adheres to the nasal mucosa and serves as a drug reservoir, prolonging the residence time of the drug in the nasal mucosa and enabling the drug to be continuously absorbed into the brain. It can be used as an excellent gel preparation for nasal drug delivery.

[0042] 2. The present invention utilizes the characteristic that the Schiff base bond formed by the aldol condensation between aldehyde group-terminated F127 and carboxymethyl chitosan is easily hydrolyzed, enabling the hydrogel to have good degradability, and the black phosphorus nanocomposite is easily degraded into non-toxic phosphates. Therefore, the hydrogel loaded with the black phosphorus-methylene blue nanocomposite of the present invention has excellent biocompatibility and is suitable for disease treatment.

[0043] 3. The preparation method of the hydrogel loaded with the black phosphorus-methylene blue nanocomposite provided by the present invention has a relatively simple production process. The solvent involved in synthesizing the composite hydrogel is water, which is non-toxic and environmentally friendly, and can achieve low-cost and large-scale production.

[0044] 4. The present invention also provides a preparation method of a black phosphorus-methylene blue nanocomposite. Methylene blue binds to black phosphorus through electrostatic adsorption and hydrophobic interaction to form a black phosphorus-methylene blue nanocomposite, improving the biodistribution of the drug; at the same time, black phosphorus nanosheets have a biological effect of scavenging reactive oxygen species and can synergistically play a neuroprotective role with methylene blue.

[0045] 5. The hydrogel loaded with the black phosphorus-methylene blue nanocomposite provided by the present invention is expected to be used as a drug delivery strategy in the multi-directional and multi-dimensional nano biomedical field, such as diseases mainly involving brain lesions in Alzheimer's disease, Parkinson's disease, stroke, etc. Brief Description of the Drawings

[0046] Figure 1 1H NMR spectrum of F127-CHO prepared in the present invention.

[0047] Figure 2 Fourier transform infrared spectra of F127-CHO and F127-CHO / CMCS hydrogels prepared in the present invention.

[0048] Figure 3 Transmission electron micrographs of freshly prepared BP NSs and BP-MB in the present invention (scale bar: 100 μm); among them, A is the transmission electron micrograph of BP NSs, and B is the transmission electron micrograph of BP-MB.

[0049] Figure 4 Atomic force micrographs of freshly prepared BP NSs and BP-MB in the present invention; among them, A is the atomic force micrograph of BP NSs, and B is the atomic force micrograph of BP-MB.

[0050] Figure 5 Fourier transform infrared spectra of BP, BP-MB, and the hydrogel loaded with the black phosphorus-methylene blue nanocomposite (BP-MB@Gel) prepared in the present invention.

[0051] Figure 6Pictures of the phase transition behavior of the F127-CHO / CMCS hydrogel and BP-MB@F127-CHO / CMCS prepared in the present invention at different temperatures.

[0052] Figure 7 Scanning electron micrographs of the F127-CHO / CMCS hydrogel and BP-MB@F127-CHO / CMCS prepared in the present invention (scale bars: 10 μm, 4 μm).

[0053] Figure 8 Results graphs of the rheological properties of the F127-CHO / CMCS hydrogel and BP-MB@F127-CHO / CMCS prepared in the present invention.

[0054] Figure 9 Results graphs of the experiment on the capture of oxygen free radicals by BP NSs and BP-MB prepared in the present invention.

[0055] Figure 10 Results graphs of the cytotoxicity experiment of BP NSs and BP-MB prepared in the present invention.

[0056] Figure 11 Results graphs of the in vitro neuroprotective effect experiment of BP NSs and BP-MB prepared in the present invention.

[0057] Figure 12 Results graphs of the in vitro oxygen consumption rate experiment of BP NSs and BP-MB prepared in the present invention.

[0058] Figure 13 Results graphs of the uptake mechanism of Cy5-labeled BP NSs on SH-SY5Y cells prepared in the present invention.

[0059] Figure 14 Results graphs of the uptake mechanism of Cy5-labeled BP NSs on HNEpC cells prepared in the present invention.

[0060] Figure 15 Results graphs of the permeability of BP-MB in HNEpC cells prepared in the present invention.

[0061] Figure 16 Results graphs of the transmembrane transport mechanism of BP-MB on HNEpC cells prepared in the present invention.

[0062] Figure 17 Fluorescence imaging graphs of Cy5-labeled BP NSs in the brain after preparation in the present invention.

[0063] Figure 18 Results graphs of the experiment on the effect of BP-MB@Gel administration on the open field behavior of animals after treatment in the present invention.

[0064] Figure 19 The figure shows the results of the Morris water maze and nesting behavior experiments of animals after treatment with the BP-MB@Gel prepared according to the present invention; among them, A-F are the results of the water maze experiment, and G-H are the results of the nesting experiment. Detailed implementation mode

[0065] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0066] Here, the abbreviations in the examples are explained: F127-CHO: aldehyde-capped triblock copolymer F127; CMCS: carboxymethyl chitosan; F127-CHO / CMCS: polymer obtained by dynamic covalent crosslinking of aldehyde-capped triblock copolymer F127-CHO with CMCS; BP NSs: black phosphorus nanosheets; MB: methylene blue; BP-MB: black phosphorus-methylene blue nanocomposite; F127-CHO / CMCS(Gel): blank hydrogel obtained by dynamic covalent crosslinking of F127-CHO with CMCS; BP-MB@F127-CHO / CMCS (BP-MB@Gel): hydrogel loaded with black phosphorus-methylene blue nanocomposite; BP-Cy5: black phosphorus nanosheets loaded with Cy5 fluorescence; BP-Cy5@Gel: hydrogel loaded with BP-Cy5.

[0067] Example 1

[0068] An embodiment of the hydrogel of the present invention. The preparation method of the hydrogel in this embodiment includes the following steps:

[0069] 1. Add 8.0 g of Pluronic to 200 mL of anhydrous dichloromethane and stir until completely dissolved. Under ice-water bath stirring, add 1.072 g of Dess-Martin reagent powder to the solution in small portions. After 30 min, heat to 40 °C and react for 24 h. Filter by suction to remove insoluble substances. Rotate evaporate the filtrate to a viscous liquid, then add it to 100 mL of ice-cold diethyl ether and stir vigorously to obtain a large amount of precipitate. Cool in an ice-water bath for 1 h. Centrifuge at 9000 rpm for 5 min, collect the precipitate, and dry it in vacuo at 40 °C for 12 h to obtain the product F127-CHO.

[0070] 2. Weigh 1 g of NaOH powder and dissolve it in 40 mL of N-methylpyrrolidone (NMP). Vortex it thoroughly for 10 - 20 min to remove the undissolved NaOH. Disperse 30 mg of black phosphorus powder in the above NMP solution saturated with NaOH. Take the above dispersion for probe sonication. Under the ice bath condition of 4 - 10 °C, sonicate it at a power of 600 W for 12 h. Then centrifuge it at 2000 rpm for 20 min, collect the supernatant. The supernatant is continued to be sonicated by probe under the above conditions and centrifuged again, with the condition of 4000 rpm for 20 min, to obtain the supernatant of BP NSs with a size of 100 - 200 nm.

[0071] 3. Add the MB aqueous solution (1.5 mL, 2 mg / mL) to the BP aqueous dispersion (1 mL, 1 mg / mL), then add 7.5 mL of deoxygenated water, stir it in the dark at room temperature for 12 h. Then centrifuge the reaction product at 12000 rpm for 15 min, wash it three times with water and resuspend it in water to obtain the BP-MB aqueous dispersion.

[0072] 4. Weigh 30 mg of F127-CHO and add it to 0.5 mL of the BP-MB dispersion (concentration of 1.5 mg / mL), dissolve it overnight at 4 °C. Weigh 40 mg of CMCS and add it to 0.5 mL of the BP-MB dispersion with the same concentration, stir until completely dissolved. Under slow stirring in an ice-water bath, mix the two solutions evenly, then place it at 37 °C and react fully for 12 h to obtain the hydrogel BP-MB@F127-CHO / CMCS (BP-MB@Gel) loaded with BP-MB.

[0073] Example 2

[0074] An example of the blank hydrogel F127-CHO / CMCS (Gel) of the present invention. The preparation method of the blank hydrogel F127-CHO / CMCS (Gel) in this example includes the following steps:

[0075] 1. Add 8.0 g of Pluronic to 200 mL of anhydrous dichloromethane and stir until completely dissolved. Under stirring in an ice-water bath, add 1.072 g of Dess-Martin reagent powder to the solution in small portions multiple times. After 30 min, heat it to 40 °C and react for 24 h. Filter it by suction to remove the insoluble substances. Rotate evaporate the filtrate into a viscous liquid, then add it to 100 mL of ice-cold diethyl ether and stir vigorously to obtain a large amount of precipitate. Cool it in an ice-water bath for 1 h. Centrifuge it at 9000 rpm for 5 min, collect the precipitate, and dry it in vacuo at 40 °C for 12 h to obtain the product F127-CHO.

[0076] 2. Weigh 30 mg of F127-CHO and add it to 0.5 mL of deionized water, and dissolve it overnight at 4 °C. Additionally, weigh 40 mg of CMCS and add it to 0.5 mL of deionized water, and stir until completely dissolved. Under slow stirring in an ice-water bath, mix the F127-CHO solution and the CMCS solution evenly. After obtaining a clear and transparent liquid, place it at 37 °C and react for 12 h to obtain the blank hydrogel F127-CHO / CMCS (Gel).

[0077] Example 3

[0078] The difference between Example 3 and Example 1 is that in step 5, 10 mg of F127-CHO is weighed and added to 0.5 mL of BP-MB dispersion (concentration: 1.5 mg / mL), and the other steps are the same.

[0079] Example 4

[0080] The difference between Example 4 and Example 1 is that in step 5, 40 mg of F127-CHO is weighed and added to 0.5 mL of BP-MB dispersion (concentration: 1.5 mg / mL), and the other steps are the same.

[0081] Example 5

[0082] Characterization and performance testing of the products prepared in Examples 1-4 of the present invention:

[0083] 1. Perform H 1 NMR characterization on the F127-CHO described in Examples 1-4. The results are as Figure 1 shown. The peak at the H 1 NMR Figure 9 .58 ppm shift represents the hydrogen in the aldehyde group, proving that F127-CHO has been successfully prepared.

[0084] 2. Perform FT-IR characterization on the F127-CHO described in Examples 1-4 and the F127-CHO / CMCS hydrogel described in Example 2. The results are as Figure 2 shown. Compared with F127, the spectrum of F127-CHO shows an absorption band for the symmetric vibration of the carbonyl group in the aldehyde group at 1727 cm -1 , indicating the successful preparation of F127 capped with aldehyde groups. In the spectrum of the F127-CHO / CMCS hydrogel (F / C Hydrogel), the characteristic absorption band of the carbonyl group at 1727 cm -1 disappears, and at the same time, there is a stretching vibration of the carboxylate anion at 1590 cm -1 , confirming the formation of a chemical bond crosslink between F127-CHO and carboxymethyl chitosan.

[0085] 3. Transmission electron microscopy tests and atomic force microscopy scans were performed on the BP NSs described in Examples 1, 3, and 4 and the BP-MB described in Example 1. As Figure 3 shown, Figure 3 A (scale bar: 100 μm) shows that the BP NSs are two-dimensional sheet structures with a lateral size of approximately 100 - 200 nm, indicating the successful exfoliation of two-dimensional black phosphorus nanosheets. Figure 3 B (scale bar: 100 μm) shows that the BP-MB has the same sheet structure and an increased size. Figure 4 A and B are the corresponding thickness data. The thickness of the BP NSs is in the range of 5 - 8 nm. After reaction with MB, the nanosheets become thinner, with a thickness of 3 - 4 nm. The BP-MB described in Examples 3 and 4 is similar to the result of Example 1.

[0086] 4. Fourier transform infrared spectroscopy (FT-IR) characterization was performed on the BP NSs described in Examples 1, 3, and 4, the BP-MB described in Example 1, and the BP-MB@Gel (drug-loaded gel). As Figure 5 shown, in the FT-IR of BP, the absorptions at 1641 cm -1 and 1001 cm -1 correspond to phosphate groups respectively; after loading MB, the obtained BP-MB shows a carbon-hydrogen bond at 1400 cm -1 , and the characteristic absorption at 3419 cm -1 indicates the existence of intermolecular hydrogen bonding in BP-MB; the spectrum of BP-MB@Gel has the characteristic absorption of BP-MB, indicating the successful loading of BP-MB in the hydrogel. The BP-MB and the BP-MB@Gel described in Examples 3 and 4 are similar to the result of Example 1.

[0087] 5. The phase transition behaviors of the F127-CHO / CMCS hydrogel described in Example 2 and the BP-MB@Gel described in Example 1 were tested. The results are as Figure 6 shown. The blank hydrogel and the hydrogel loaded with BP-MB are in a fluid state at room temperature. When the temperature reaches 37 °C, both undergo sol-gel phase transitions, confirming their temperature responsiveness. Scanning electron microscopy tests were performed on the F127-CHO / CMCS hydrogel described in Example 2 and the BP-MB@Gel described in Example 1. Figure 7 It shows that both present a uniform porous network structure, and the loading of BP-MB does not damage the gel structure. The BP-MB@Gel described in Examples 3 and 4 is similar to the result of Example 1.

[0088] 6. Rheological property tests were performed on the F127-CHO / CMCS hydrogel described in Example 2 and the BP-MB@Gel described in Example 1. As Figure 8As shown in Figure A, as the temperature increases, the storage modulus G’ of the gel is higher than the loss modulus G”. The temperature at which G’ = G” is the sol-gel phase transition temperature. The phase transition temperatures of the blank gel and the drug-loaded gel are 29.8 °C and 31.8 °C respectively, confirming that it can form a gel under body temperature conditions. Oscillatory shear and stress tests were carried out on the F127-CHO / CMCS hydrogel described in Example 2 and the BP-MB@Gel described in Example 1 to investigate the mechanical properties of the gel. The results of the frequency sweep test are as Figure 8 shown in Figure B. Both G’ and G” increase with the increase of the angular frequency, and G’ is always greater than G”, indicating the formation of a solid gel. The stress test is as Figure 8 shown in Figure C. When the applied stress is greater than 160%, the blank gel and the drug-loaded gel can be transformed from a solid gel to a fluid. As can be seen from Figure 8 Figure D, as the shear rate increases, the viscosity of the drug-loaded gel decreases, proving its shear-thinning property, so it can be used for injection. The self-healing hydrogel constructed based on covalent bonds undergoes dynamic changes in modulus or viscosity when subjected to reciprocally changing shear strain and can return to its original state. Figure 8 Figures E and 8F are the high and low strain sweep diagrams of the blank gel and the drug-loaded gel respectively. Under the action of alternating stress of 1% and 500%, the modulus of the drug-loaded gel can return to the value at a smaller strain, proving its good self-healing ability. The results of BP-MB@Gel described in Examples 3 and 4 are similar to those of Example 1.

[0089] 7. Antioxidant performance tests were carried out on the BP NSs described in Examples 1, 3, and 4 and the BP-MB described in Example 1. As Figure 9 shown, the BP NSs described in Examples 1, 3, and 4, the BP-MB described in Example 1, and the drug MB have certain scavenging abilities for different free radicals such as hydroxyl radicals (·OH), superoxide anions (O 2 ·- ,), and hydrogen peroxide (H 2 O 2 ). Their antioxidant abilities show a dose-dependent relationship, and due to the loading of the antioxidant MB, BP-MB has a stronger ability to scavenge free radicals. The results of BP-MB described in Examples 3 and 4 are similar to those of Example 1.

[0090] Example 6

[0091] An example of the in vitro safety evaluation experiment of BP-MB described in Example 1 of the present invention. The in vitro safety evaluation experiment of BP-MB described in this example includes the safety evaluation of BP-MB at the levels of human neuroblastoma (SH-SY5Y) cells and human nasal mucosa epithelial cells (HNEpC).

[0092] The SH-SY5Y cells and HNEpC cells were seeded into 96-well plates at a density of 3×10 4 cells / well and 1×10 4 / well, respectively. After culturing for 24 h, gradient concentrations of MB, BP, and BP-MB were added and co-incubated, and then the cells were cultured for another 24 h. Subsequently, CCK-8 solution was added and incubated for 4 h, and a microplate reader was used to measure the absorbance at 450 nm. Cell viability was expressed as the percentage of the drug-treated group relative to the blank control group at A450nm. The results are shown in Figure 10 Figure A. After incubation with SH-SY5Y for 24 h, there was no significant change in cell viability when the concentration ranges of MB were 1-8 μg / mL, and those of BP and BP-MB were 1.25-10 μg / mL. As shown in Figure 10 Figure B, when the concentration ranges of MB were 1-10 μg / mL, and those of BP and BP-MB were 2.5-40 μg / mL, there was no significant difference in the viability of HNEpC cells compared with the control group, indicating good biocompatibility.

[0093] Example 7

[0094] Verification of the neuroprotective effect of BP-MB described in Example 1 of the present invention at the SH-SY5Y cell level and determination of mitochondrial oxygen consumption at the SH-SY5Y cell level:

[0095] 1. Verification of the neuroprotective effect of BP-MB at the SH-SY5Y cell level: SH-SY5Y cells were seeded into 96-well plates at a density of 3×10 4 cells / well. After 24 h, the cells were pretreated with 40 nM okadaic acid for 12 h, and then the medium was replaced. Gradient concentrations of MB, BP, and BP-MB were added, and after culturing for another 24 h, CCK-8 solution was added. After 4 h, a microplate reader was used to measure the absorbance value at 450 nm. Cell viability was expressed as the percentage of the drug-treated group relative to the blank control group at A450nm. The results are shown in Figure 11 Figure. In the concentration range of 1.25-10 μg / mL, compared with the free MB group and BP group, BP-MB showed a more excellent in vitro neuroprotective effect (P<0.05), and cell viability increased in a dose-dependent manner.

[0096] 2. Determination of mitochondrial oxygen consumption of BP-MB at the SH-SY5Y cell level: SH-SY5Y cells (3×10 4Cultured for 24 h on a 96-well plate (number of cells / well), and then 40 nM okadaic acid was added and incubated for another 12 h. Subsequently, the medium was replaced, and 2 μg / mL of MB, 10 μg / mL of BP, and 10 μg / mL of BP-MB were added respectively and incubated for 24 h. Before performing the mitochondrial stress test, the cell medium was replaced with FX medium (175 μL). After the cells were equilibrated at 37 °C for 60 min, the mitochondrial stress test was carried out by adding a mixture of mitochondrial inhibitors: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM), oligomycin A (1 μM), antimycin A (1 μM), and rotenone (1 μM). The results are as Figure 12 shown. After treatment with BP-MB, the mitochondrial dysfunction caused by okadaic acid was significantly improved, and the levels of energy indicators such as basal respiration, ATP production, H + proton leakage, and maximal respiration were increased. Compared with the MB and BP groups, it had a more excellent effect of reversing mitochondrial dysfunction (P < 0.05).

[0097] The above results indicate that BP-MB has better biocompatibility and has a more obvious neuroprotective effect than free MB and BP, and can improve the mitochondrial disorder in the in vitro AD model.

[0098] Example 8

[0099] The preparation method of BP-Cy5 in this example includes the following steps:

[0100] Add the aqueous solution of Cy5-PEG-NH 2 (2.5 mL, 2 mg / mL) to the aqueous dispersion of BP (1 mL, 1 mg / mL), add 1.5 mL of deoxygenated water, sonicate in an ice-water bath (10 °C) for 30 min in the dark, stir at room temperature for 8 h, then centrifuge the reaction product at 12000 rmp for 15 min, wash the substrate three times with water and resuspend with water to obtain the BP-Cy5 dispersion loaded with Cy5.

[0101] The uptake mechanism investigation experiment of BP-Cy5 by SH-SY5Y cells and HNEpC cells was carried out using the BP-Cy5 dispersion:

[0102] After SH-SY5Y cells and HNEpC cells grew to an appropriate density, the medium was replaced with a medium containing MβCD (methyl-β-cyclodextrin), CPZ (chlorpromazine), HS (hypertonic sucrose), or EIPA (5-(N-ethyl-N-isopropyl)-amiloride), and the cells were cultured at 37 °C for 30 min. Subsequently, BP-Cy5 (retaining inhibitor) was added, and the well plate was incubated at 37 °C in the dark for 2 h. Then, the medium was discarded, and the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 15 - 20 min. DAPI staining solution was added, and the cells were continuously cultured in the dark for 10 min. The staining solution was discarded, and the cells were washed 3 times with PBS, then sealed, and the cell uptake was observed under a laser confocal microscope. The results are as Figure 13 and 14 shown. Both SH-SY5Y cells and HNEpC cells mainly take up BP-Cy5 through the pathways mediated by clathrin and macropinocytosis. Among them, SH-SY5Y cells mainly take up BP-Cy5 through clathrin-mediated endocytosis, and HNEpC cells mainly take up BP-Cy5 through macropinocytosis-mediated endocytosis.

[0103] Example 9

[0104] Transnasal mucosal barrier experiment of BP-MB described in Example 1 of the present invention at the HNEpC cell level:

[0105] HNEpC cells were seeded at a density of 5×10 5 / cm 2 on the upper chamber of Transwell to form a monolayer of dense cells. When the transmembrane resistance value > 200 Ω·cm 2 , it indicates that the establishment of a complete nasal mucosal barrier is successful and can be used to verify the permeability of drugs across the nasal mucosa in vitro. To ensure the integrity of the in vitro nasal mucosal barrier, the TEER value was measured before and after the experiment. To measure the apical-to-basolateral transepithelial transport, 300 μL of DMEM solutions containing MB (8 μg / mL) and BP-MB (equal dose of MB) were respectively added to the apical side (upper compartment), and the cells were incubated at 37 °C for 2 h. Subsequently, the solution in the upper chamber was collected, and the concentration of MB was detected by an ultraviolet spectrophotometer. The permeability was calculated using the following formula: Permeability (%) = (C0 - Cs) / C0 × 100%, where C0 and Cs are the concentrations of the drug in the upper chamber before and after incubation, respectively. The results are as Figure 15 shown in A. Both MB and BP-MB showed a permeability as high as about 65%. As shown in Figure 14 B, and there was no significant change in the membrane potential of the nasal mucosal cell layer before and after treatment, indicating that the transport of MB and BP-MB did not affect the integrity of the nasal mucosal barrier.

[0106] Example 10

[0107] Investigation experiment on the transport mechanism of BP-MB described in Example 1 of the present invention:

[0108] HNEpC cells were seeded in the upper chamber of Transwell at a density of 5×10 5 / cm 2 . When the transmembrane resistance value > 200 Ω·cm 2 , the upper chamber was respectively added with 0.3 mL of medium containing 32.5 μg / mL of Monensin (inhibiting the transport from Golgi apparatus to plasma membrane), 25 μg / mL of Brefeldin A (specifically blocking the transport of vesicles from endoplasmic reticulum to Golgi apparatus) and 2.5 μg / mL of Bafilomycin (inhibiting endocytic acidification) and co-incubated for 1 h. The control group was added with an equal volume of DMEM, and the lower chamber was 1 mL of DMEM. Subsequently, BP-MB (retaining inhibitors except Monensin and Bafilomycin) was added and incubated for 2 h, and then the solution in the lower chamber was collected. The concentration of BP-MB in the lower chamber was measured by ultraviolet spectrophotometer, and the relative transport rate of each group was calculated by comparing with the control group. As shown in Figure 16 A, different protein transport inhibitors at corresponding concentrations did not produce toxicity to cells after co-incubation with HNEpC for 24 h. The results are as shown in Figure 16 B. Monensin and Brefeldin A significantly inhibited the transport of BP-MB, indicating that the transport pathway of BP-MB across nasal mucosal epithelial cells mainly involves the transport from Golgi apparatus to plasma membrane and from endoplasmic reticulum to Golgi apparatus.

[0109] Example 11

[0110] BP-Cy5 loaded with Cy5 fluorescence was prepared according to the steps described in Example 8. The preparation method of the hydrogel loaded with BP-Cy5 in this example includes the following steps:

[0111] 30 mg of F127-CHO was added to 0.5 mL of BP-Cy5 dispersion (concentration: 1.5 mg / mL) and dissolved overnight at 4°C. 40 mg of CMCS was added to 0.5 mL of BP-Cy5 dispersion with the same concentration and stirred until completely dissolved. The two solutions were mixed evenly under slow stirring in an ice-water bath and then placed at 37°C for full reaction for 12 h to obtain the hydrogel loaded with BP-Cy5 (BP-Cy5@Gel).

[0112] The brain targeting characteristics of the nasal administration gel preparation were studied using the said BP-Cy5 and the said BP-Cy5@Gel.

[0113] Female C57BL / 6 mice were randomly divided into 3 groups: ① Cy5 group, ② BP-Cy5 group, ③ BP-Cy5@Gel group (n = 3). Each group of mice was given an equal dose of Cy5 through nasal cavity, and whole brains were taken at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h for fluorescence imaging under a live imaging instrument. The results are as shown in Figure 17As shown, after 4 h of administration, free Cy5 was gradually metabolized in the brain, while the accumulation of BP-Cy5@Gel in the brain increased, reaching the maximum cumulative amount at 8 h, and there was still a small amount of accumulation in the brain after 24 h. In addition, in the imaging at each time point, the BP-Cy5@Gel group showed strong fluorescence distribution. These results indicate that intranasal administration of BP-Cy5@Gel can significantly increase the drug accumulation in the brain, and the gel adhering to the nasal cavity can slowly release the drug, improving the bioavailability of the drug in the brain, thus achieving good therapeutic effects.

[0114] Example 12

[0115] Using the BP-MB@Gel described in Example 1 of the present invention to study the pharmacodynamics of the intranasal administration gel preparation against Alzheimer's disease in vivo:

[0116] Six-month-old female wild-type C57BL / 6 mice were used. Mice in each group were fasted for 24 h before surgery without water deprivation. After intraperitoneal anesthesia, the mice were fixed on a small animal stereotaxic apparatus. The scalp of the mice was cut open, and after exposing the skull, it was disinfected with 75% alcohol. According to the mouse brain stereotaxic atlas, a hole was drilled in the lateral amygdala (posterior to bregma: -1.94 mm, lateral to midline: -3.15 mm). OA was dissolved in DMSO and diluted with normal saline. 130 nL of the diluted 100 μM OA solution was precisely aspirated and injected into the lateral amygdala, with the injection depth of -4.5 mm, the injection speed maintained at 60 nL / min, the injection lasting for 5 min, and the injection needle was kept in place for 5 min after injection to ensure complete infusion of the drug, and then the injection needle was slowly withdrawn from the mouse brain. After that, penicillin powder was covered on the mouse brain drilling site to prevent infection and death, and the scalp was sutured with surgical thread. Mice were normally given water and food, and drug administration treatment started three days later. The sham operation group was injected with an equal volume of 0.9% normal saline.

[0117] The model mice were randomly divided into four groups for drug administration treatment: ① MB; ② BP; ③ BP-MB; ④ BP-MB@Gel (equal BP dose: 3 mg / kg, equal MB dose: 0.6 mg / kg), the administration volume was 30 μL, once a day for seven consecutive days. At the same time, the OA model group was given an equal volume of normal saline. After the drug administration ended, a behavioral evaluation experiment was carried out, and the behavioral evaluation experiment included an open field test, a water maze test, and a nesting test.

[0118] Open field test: The mice were placed in an activity empty box (60 cm × 60 cm × 40 cm). Before the test, the mice were placed in the center of the empty box and allowed to freely explore for 10 minutes to adapt in advance. Then the total moving distance and average moving speed were recorded during the 20-minute test. The results are as Figure 18 shown.

[0119] Morris water maze experiment: The MWM setup consisted of a circular pool (150 cm in diameter and 50 cm in height). White powder was added to the water in the circular pool to make the black C57BL / 6 mice easier to track. Before learning and training, the mice were placed in the water maze for 2 min of free swimming for adaptation. During training, the mice were randomly placed into the water facing the pool wall starting from the northwest, southwest, or northeast direction. The maximum swimming time was set to 90 s to find the hidden platform. If the platform was successfully found, the time to find the hidden platform, i.e., the escape latency, was recorded, and the mice were allowed to stay on the platform for 15 s. If the platform was not found within 90 s, the mice were guided to the platform and allowed to stay for 15 s. After swimming, the mice were carefully dried and baked with clothes. One direction was defined as one round. Each mouse was trained 3 rounds per day, with an interval of 1 h between each training session, and trained continuously for 4 days, with different directions for training each day. On the 5th day, a spatial location exploration experiment was conducted. The hidden escape platform was removed, and the mice were placed into the water facing the pool wall from the opposite quadrant (northwest) of the hidden platform, and the number of times and the staying time of the mice crossing the original position of the hidden platform within 60 s were recorded. The results are as Figure 19 shown in A - F.

[0120] Nest - building experiment: Before the nest - building test, the mice were individually housed with a supply of corncobs for one week. On the first day, 6 pieces of paper (5×5 cm 2 ) were placed into the cage to provide conditions for the mice to build nests. The specific scoring criteria for the quality of the nest (from low to high) were as follows: 1 to 4: 1. The paper had no obvious biting / tearing and no recognizable nest site; 2. There was no obvious biting / tearing of the paper, and there was a recognizable nest site; 3. The paper was partially bitten / ripped, and there was a recognizable nest site; 4. The most severely bitten / ripped paper with a recognizable nest. The nest - building situation of each group of mice was observed on the third day. The results are as Figure 19 shown in G - H.

[0121] The results of behavioral experiments showed that: In the open - field experiment, the spontaneous activity ability and spatial exploration ability of the mice treated with BP - MB@Gel were significantly enhanced; After five days of spatial learning in the water maze, the learning and memory ability of the mice in the BP - MB@Gel treatment group was significantly improved; In the nest - building experiment, due to the learning and memory ability defects, the AD mice could not build a complete nest, while the nest - building behavior of the mice in the BP - MB@Gel treatment group was significantly improved. These results indicate that the prepared nasal - administration gel preparation has the effect of improving the behavioral and cognitive dysfunction of AD mice.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but they do not deviate from the essence and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a hydrogel, characterized in that, it includes the following steps: (1) Add aldehyde-terminated poly(ethylene glycol) 106 -poly(propylene glycol) 70 -poly(ethylene glycol) 106 The triblock polymer F127-CHO was added to dispersion A of black phosphorus nanocomposite loaded with methylene blue and dissolved to obtain mixture A; (2) Add carboxymethyl chitosan to dispersion B of black phosphorus nanocomposite loaded with methylene blue, dissolve it to obtain mixture B; (3) Mix mixture A and mixture B evenly, and obtain the hydrogel after sufficient reaction; The concentration of the black phosphorus nanocomposite loaded with methylene blue in dispersion A of the black phosphorus nanocomposite loaded with methylene blue is the same as that in dispersion B of the black phosphorus nanocomposite loaded with methylene blue; The mass ratio of F127-CHO to carboxymethyl chitosan is (1-4):4; The preparation method of the black phosphorus nanocomposite loaded with methylene blue includes the following steps: S1. Add an aqueous solution of methylene blue to the aqueous dispersion of black phosphorus nanosheets, and react under a state of stirring in the dark to obtain a reaction product; S2. Centrifuge the obtained reaction product, wash it with an aqueous solution, and then resuspend it in water to obtain a dispersion of the black phosphorus nanocomposite loaded with methylene blue; In the above step S1, the concentration of black phosphorus nanosheets in the aqueous dispersion of black phosphorus nanosheets is 0.5-2 mg / mL, and the concentration of methylene blue in the aqueous solution of methylene blue is 1-3 mg / mL; the volume ratio of the aqueous dispersion of black phosphorus nanosheets to the aqueous solution of methylene blue is 1:(1-3).

2. The preparation method of the hydrogel as described in claim 1, characterized in that, the mass ratio of F127-CHO to carboxymethyl chitosan is 3:

4.

3. The preparation method of the hydrogel as described in claim 1, characterized in that, in dispersion A of the black phosphorus nanocomposite loaded with methylene blue and dispersion B of the black phosphorus nanocomposite loaded with methylene blue, the concentration of the black phosphorus nanocomposite loaded with methylene blue is 0.1-3 mg / mL.

4. The preparation method of the hydrogel as described in claim 1, characterized in that, the particle size of the black phosphorus nanosheets is 100-200 nm, and the thickness is 5-8 nm.

5. The preparation method of the hydrogel as described in claim 1, characterized in that, the preparation method of F127-CHO includes the following steps: S1. Weigh Pluronic and dissolve it in an organic solvent, add Dess-Martin oxidant, stir and heat for an oxidation reaction; S2. Take the filtrate obtained from the oxidation reaction in step S1, concentrate it to a viscous state, then add it to ice ether and stir to produce a precipitate, cool it, and centrifuge to collect the precipitate to obtain F127-CHO.

6. A hydrogel prepared by the preparation method of the hydrogel as described in any one of claims 1-5.

7. The application of the hydrogel as described in claim 6 in the preparation of a drug for treating neurodegenerative diseases, characterized in that, the neurodegenerative disease is Alzheimer's disease.

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