A porous gel scaffold for zoned drug delivery, its preparation method and application

Abstract

This invention relates to the field of materials science and technology, providing a porous gel scaffold for partitioned drug delivery, its preparation method, and its applications. The modification steps in this invention are simple, allowing for easy acquisition of the target product. The purification method is straightforward, and the modified monomers exhibit good biocompatibility and high safety. This invention modifies hyaluronic acid with norbornene and gelatin with thiol groups, effectively reducing its liquid viscosity and facilitating emulsification in liquid microfluidic chips. The synthesized microgels exhibit high uniformity in particle size, strong controllability, and stable drug loading concentration in a single gel. With the assistance of photocurable cyclodextrin, the aqueous solubility of non-water-soluble drug molecules can be significantly improved. The porous gel scaffold prepared by secondary assembly of the microgels used in this invention can better simulate the three-dimensional environment in vivo and can be used as a modular tissue engineering repair material for regenerative medicine drug / gene targeted delivery and stem cell loading.

Description

Technical Field

[0001] This invention belongs to the field of materials science and technology, and particularly relates to a zoned drug delivery porous gel scaffold, its preparation method and application. Background Technology

[0002] Osteoarthritis (OA) is a widespread degenerative disease. Studies have shown that cartilage-bone interface defects in OA caused by trauma, disease, or aging typically involve damage to the hyaline cartilage and subchondral bone. When both cartilage and subchondral bone are damaged simultaneously, the lack of nutrient delivery channels such as blood vessels, nerves, and lymphatic vessels in the cartilage itself leads to poor cartilage regeneration. The clinical challenge of effectively filling tissue defects while simultaneously promoting bone and cartilage regeneration and vertically integrating the bone-cartilage interface remains unresolved. Traditional clinical treatments for OA primarily focus on repairing the cartilage layer, with treatment options including microfractures, autologous cartilage transplantation, allogeneic transplantation, and autologous cell transplantation. However, these methods suffer from numerous problems, such as limited donors, restricted cartilage regeneration, and post-regeneration bone-cartilage delamination, often leading to surgical failure or multiple surgeries, increasing patient suffering and financial burden. Therefore, developing an effective treatment to promote regeneration in the joint defect area and reshape the bone-cartilage interface is essential.

[0003] Hydrogels are hydrophilic polymers with high water content. They offer diverse processing methods, are similar to the extracellular matrix (ECM), and their physicochemical and biological properties can be adjusted as needed, making them a highly promising tissue engineering material in the life sciences. Hydrogels stabilize water molecules within their bulk structure through their own hydrophilic network, creating a barrier between the functional factors they carry and the surrounding tissues. This affects the interaction between the functional factors and the surrounding tissues, reducing therapeutic efficacy. Furthermore, most bulk gels currently available have poor compatibility with damaged tissue areas; smaller volumes tend to create cavities, while larger volumes can compress surrounding tissues and cause edema. Processing bulk gels into microgels with diameters ranging from 1 to 1000 mm effectively addresses these issues. Microgels possess shear-thinning properties, allowing for delivery via needle extrusion, facilitating the filling of irregular wounds. They can also be used for secondary polymerization and assembly into porous structures, improving factor diffusion efficiency and the metabolic rate of the tissue microenvironment. Summary of the Invention

[0004] The purpose of this invention is to provide a zoned drug delivery porous gel scaffold, its preparation method, and its application, aiming to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing a zoned drug delivery porous gel scaffold includes the following steps:

[0007] Step S1: Preparation of norbornene-modified hyaluronic acid: Dissolve 1-3 g of hyaluronic acid in deionized water. After complete dissolution, add 15-20 g of adipic acid dihydrazide and 3-5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Maintain the pH at 4.5-5 during the reaction. After 8-12 h of reaction, pour the solution into a dialysis bag and dialysis for 3-5 days. Freeze-dry the dialysis solution to obtain adipic acid dihydrazide-modified hyaluronic acid. Alternatively, dissolve 1-3 g of adipic acid dihydrazide-modified hyaluronic acid in deionized water. Dissolve 4-5 g of cis-5-norbornene-endo-2,3-dicarboxylic anhydride in 80-120 mL of dimethylformamide and add it dropwise to the adipic acid dihydrazide-modified hyaluronic acid solution. Maintain the pH at 6.5-7.5 during the reaction. After 8-12 h of reaction, pour the solution into a dialysis bag and dialysis for 3-5 days. d. After freeze-drying the dialysis solution, norbornene-modified hyaluronic acid was obtained and stored at -90 to -70 °C.

[0008] Step S2: Preparation of thiol-modified gelatin: Dissolve 1-2 g of dimethyl 3,3-dithiodipropionate and 1-2 mL of hydrazine hydrate in 20-30 mL of methanol, react for 8-12 h, filter the suspension, and wash the resulting white solid with methanol and deionized water for later use; dissolve 1-2 g of gelatin in deionized water, add 0.3-1 g of the aforementioned white solid and 0.1-1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, react for 1-3 h, adjust the pH of the solution to 7, and terminate the reaction; add 2-3 g of dithiothreitol to the solution, react for 20-30 h, adjust the pH of the solution to 8.5, and then adjust the pH of the solution to 3-4; pour the reaction solution into a dialysis bag and dialyze in hydrochloric acid solution with pH=4.5-5 for 1-3 hours. d, then dialysis in deionized water for 1~2 days, freeze-dry the dialysis solution to obtain mercapto-modified gelatin, and store at -90~-70 ℃;

[0009] Step S3: Preparation of double-bond modified cyclodextrin: Dissolve 8-12 g of cyclodextrin in 100-200 mL of dimethylformamide, add 3-4 mL of triethylamine and 2-3 mL of acryloyl chloride dropwise to the solution, react for 8-12 h, filter to remove solids, pour the resulting liquid into a dialysis bag and dialysis for 3-5 days, freeze dry to obtain double-bond modified cyclodextrin, and store at -90 to -70 ℃.

[0010] Step S4: Prepare a droplet microfluidic chip; the droplet microfluidic chip includes an aqueous phase inlet 1, an aqueous phase inlet 2, an oil phase inlet, and a droplet outlet;

[0011] Step S5: Dissolve the norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, photoinitiator 2959, and bergenin in PBS solution at mass concentrations of 10–20 mg / mL, 50–100 mg / mL, 5–10 mg / mL, and 3–4 × 10⁻⁶ mg / mL, respectively. -4 A solution of 50-100 mg / mL of thiol-modified gelatin obtained in step S2 is introduced into the aqueous phase through inlet 1 at a flow rate of 75-200 μL / h. This solution is then used as the aqueous phase through inlet 2. Mineral oil containing SPAN80 is introduced into the oil phase through inlet 400-1000 μL / h. After the liquid is introduced, the aqueous and oil phases meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out through the droplet transport channels from the droplet outlet. The resulting microgel prepolymer microspheres are exposed to ultraviolet light for 3-5 min, and then washed sequentially with mineral oil, a PBS solution containing TWEEN 20, and another PBS solution to obtain a microgel loaded with bergenin.

[0012] The norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, and photoinitiator 2959 were dissolved in PBS solution at concentrations of 10–20 mg / mL, 50–100 mg / mL, and 5–10 mg / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 1 at a flow rate of 75–200 μL / h. The thiol-modified gelatin and bone morphogenetic protein obtained in step S2 were dissolved in PBS solution at concentrations of 50–100 mg / mL and 80–120 ng / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 2. Mineral oil containing SPAN80 was introduced as the oil phase through the oil phase inlet at a flow rate of 400–1000 μL / h. μL / h; After the liquid is introduced, the aqueous phase and the oil phase meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out from the droplet outlet through the droplet transport channels. The obtained microgel prepolymer microspheres are exposed to ultraviolet light for 3 min to 5 min, and then washed sequentially with mineral oil, PBS solution containing TWEEN 20, and PBS solution to obtain microgels carrying bone morphogenetic proteins.

[0013] Step S6: The microgels loaded with bergenin and bone morphogenetic proteins obtained in step S5 are stacked according to the preset cartilage injury area. After being soaked in the thiol-modified gelatin aqueous solution obtained in step S2, excess liquid is removed. After a second UV exposure for 15-30 s, a zoned drug delivery porous gel scaffold is obtained.

[0014] Furthermore, in step S1, the molecular weight of hyaluronic acid is 9~12 w.

[0015] Furthermore, in step S2, the gelatin is pigskin gelatin or fish skin gelatin, and the dialysis bag material in both steps S2 and S1 is cellulose with a molecular weight cutoff of 8000~15000 Da.

[0016] Furthermore, in step S3, the cyclodextrin is β-cyclodextrin, the dialysis belt is made of cellulose, and the molecular weight cutoff is 300~500 Da.

[0017] Furthermore, in step S5, the mass fraction of SPAN80 in the mineral oil is 10%~20%.

[0018] Furthermore, in step S6, the mass fraction of thiol-modified gelatin is 50-100 mg / mL, and the aqueous solution of thiol-modified gelatin contains 5-10 mg / mL of photoinitiator 2959.

[0019] A partitioned drug delivery porous gel scaffold prepared according to a preparation method.

[0020] Application of a zoned drug delivery porous gel scaffold in the preparation of drugs for treating osteoarthritis injuries.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. The modification process of the material in this invention is simple, the target product can be obtained through simple operation, the purification method is simple, and the modified monomer has good biocompatibility and high safety.

[0023] 2. This invention modifies hyaluronic acid with norbornene and gelatin with thiol groups, which can effectively reduce its liquid viscosity and facilitate emulsification operation in liquid microfluidic chips. The synthesized microgels have high uniformity in particle size, strong controllability, and stable drug loading concentration in a single gel. With the assistance of photocurable cyclodextrin, the aqueous solubility of non-water-soluble drug molecules can be significantly improved.

[0024] 3. The porous gel scaffold prepared by the secondary assembly of microgels used in this invention can better simulate the three-dimensional environment in vivo and can be used as a modular tissue engineering repair material in the fields of regenerative medicine drug / gene targeted delivery and stem cell loading. Attached Figure Description

[0025] Figure 1In the image, (a) is a schematic diagram of the droplet microfluidic chip structure; (b) is a diagram of the microfluidic droplet chip microsphere production process; (c) is a photograph of the microgel in the oil phase after UV-induced polymerization; and (d) is a bar chart of the microgel diameter distribution at different aqueous and oil phase flow rates (the horizontal axis is the oil phase flow rate, the vertical axis is the microgel particle size, and the upper right corner values ​​25, 50, 100, and 200 represent aqueous phase flow rates of 25 μL / h, 50 μL / h, 100 μL / h, and 200 μL / h, respectively).

[0026] Figure 2 In the image, (a) is the NMR spectrum of thiol-modified gelatin; (b) is the NMR spectrum of norbornene-modified hyaluronic acid; and (c) is the NMR spectrum of double-bond-modified cyclodextrin. GelSH represents thiol-modified gelatin, Gel represents unmodified gelatin, HANB represents norbornene-modified hyaluronic acid, HA represents unmodified hyaluronic acid, CDMA represents double-bond-modified cyclodextrin, and CD represents unmodified cyclodextrin. The arrows on the spectra indicate the characteristic peak positions of the modified functional groups.

[0027] Figure 3 In the image, (a) is a schematic diagram of a syringe extruding a microgel; (b) is an image of a 3D-printed microgel; and (c) is a schematic diagram of a syringe needle extruding a porous gel scaffold for zoned drug delivery.

[0028] Figure 4 Gross images of rat joint injuries (Sham: untreated joint injury group; Control: control group, untreated joint injury group; SHNB: porous gel scaffold group, without drug loading; BK-Solution: bergenin and bone morphogenetic protein solution group, bergenin concentration 3.2×10⁻⁶). -4 mg / mL, bone morphogenetic protein concentration of 100 ng / mL, saturation administration, excess droplets wiped off with gauze; BK-SHNB: partitioned dual-drug-loaded gel scaffold assembly, the lower layer is a microgel loaded with bone morphogenetic protein at a concentration of 100 ng / mL, with a filling thickness of 1 mm, and the upper layer is loaded with bergenin at a concentration of 3.2 × 10 mg / mL. -4 The microgel was filled with a thickness of 1 mm and a concentration of mg / mL. After filling with a double layer of microgel, the obtained thiol-modified gelatin aqueous solution was added for wetting (the mass fraction of thiol-modified gelatin was 50 mg / mL, containing 5 mg / mL of photoinitiator 2959). After removing the excess liquid, the microgel was exposed to UV light for 30 s to obtain a zoned drug delivery porous gel scaffold (scale bar: 5 mm).

[0029] Figure 5Micro-CT images of rat joint injuries (Sham: untreated joint injury group; Control: control group, untreated joint injury group; SHNB: porous gel scaffold group, without drug loading; BK-Solution: bergenin and bone morphogenetic protein solution group, bergenin concentration 3.2×10⁻⁶). -4 mg / mL, bone morphogenetic protein concentration of 100 ng / mL, saturation administration, excess droplets wiped off with gauze; BK-SHNB: partitioned dual-drug-loaded gel scaffold assembly, the lower layer is a microgel loaded with bone morphogenetic protein at a concentration of 100 ng / mL, with a filling thickness of 1 mm, and the upper layer is loaded with bergenin at a concentration of 3.2 × 10 mg / mL. -4 The microgel was filled with a thickness of 1 mm and a concentration of mg / mL. After filling with a double layer of microgel, the obtained thiol-modified gelatin aqueous solution was added for wetting (the mass fraction of thiol-modified gelatin was 50 mg / mL, containing 5 mg / mL of photoinitiator 2959). After removing the excess liquid, the microgel was exposed to UV light for 30 s to obtain a zoned drug delivery porous gel scaffold (scale bar: 5 mm). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0032] An embodiment of the present invention provides a method for preparing a partitioned drug delivery porous gel scaffold, comprising the following steps:

[0033] Step S1: Preparation of norbornene-modified hyaluronic acid: Dissolve 1-3 g of hyaluronic acid in 300-500 mL of deionized water. After complete dissolution, add 15-20 g of adipic acid dihydrazide and 3-5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Adjust the pH to 4.5-5 with 1 mol / L HCl aqueous solution throughout the process. After reacting for 8-12 h, pour the solution into a dialysis bag and dialyze for 3-5 days. Freeze-dry the dialysis solution to obtain adipic acid dihydrazide-modified hyaluronic acid. Dissolve 1-3 g of adipic acid dihydrazide-modified hyaluronic acid in 300-500 mL of deionized water. Dissolve 4-5 g of cis-5-norbornene-endo-2,3-dicarboxylic acid anhydride in 80-120 mL of dimethylformamide. Add the solution dropwise to the adipic acid dihydrazide-modified hyaluronic acid solution. Adjust the pH of the solution to 6.5-7.5 with 1 mol / L NaOH aqueous solution throughout the process. After reacting for 8-12 h, pour the solution into a dialysis bag and dialysis for 3-5 days. Freeze-dry the dialysis solution to obtain norbornene-modified hyaluronic acid.

[0034] Store at -70 ℃;

[0035] Step S2: Preparation of thiol-modified gelatin: Dissolve 1-2 g of dimethyl 3,3-dithiodipropionate and 1-2 mL of hydrazine hydrate in 20-30 mL of methanol. React for 8-12 h, filter the suspension, and wash the resulting white solid thoroughly with methanol and deionized water. Dissolve 1-2 g of gelatin in 100-200 mL of deionized water (50 °C), add 0.3-1 g of the above white solid and 0.1-1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, react for 1-3 h, and then adjust the pH of the solution to 7 with 1 mol / L NaOH aqueous solution to terminate the reaction. Add 2-3 g of dithiothreitol to the above solution, react for 20-30 h, and then adjust the pH of the solution to 8.5 with 1 mol / L NaOH aqueous solution, and then adjust the pH of the solution to 3-4 with 1 mol / L HCl aqueous solution. The above reaction solution was poured into a dialysis bag and dialyzed in hydrochloric acid solution with pH=4.5~5 for 1~3 days, and then dialyzed in deionized water for 1~2 days. The dialysis solution was freeze-dried to obtain mercapto-modified gelatin, which was stored at -90~-70 ℃.

[0036] Step S3: Preparation of double-bond modified cyclodextrin: Dissolve 8-12 g of cyclodextrin in 100-200 mL of dimethylformamide, add 3-4 mL of triethylamine and 2-3 mL of acryloyl chloride dropwise to the solution, react for 8-12 h, filter to remove solids, pour the resulting liquid into a dialysis bag and dialysis for 3-5 days, freeze dry to obtain double-bond modified cyclodextrin, and store at -90 to -70 ℃.

[0037] Step S4: Fabrication of the droplet microfluidic chip: The droplet microfluidic chip is fabricated through substrate processing, spin-coating of photoresist, exposure using a UV mask, plasma etching, polydimethylsiloxane (PDMS) molding, and channel bonding, as detailed in [David A. Weitz et al. Small, 1702955, 14, (2018)]. The channel structure consists of two parallel aqueous phase inlets and aqueous phase transport channels (straight channels with a width of 100-200 μm and a length of 6-8 mm), an oil phase inlet and two symmetrically distributed oil phase transport channels (zigzag channels with a width of 100-200 μm and a length of 2-3 cm) on both sides of the aqueous phase transport channels, with a maximum parallel distance of 6-8 mm from the aqueous phase transport channels; a droplet outlet and droplet transport channels (sinusoidal channels with a width of 300-500 μm and a length of 3-4 cm; this structure is beneficial for droplet stability; sinusoidal curve) The specific parameters of the equation have little impact on the droplet preparation results. As long as the wave-shaped pattern is similar, it can stabilize the sphere and reduce the impact of the sphere behind on the sphere in front. The droplet is composed of three parts: the distance between the water phase inlet and the oil phase inlet is 8~10mm, the distance between the water phase inlet and the droplet outlet is 2~3cm, the angle between the intersection of the single oil phase transport channel and the water phase transport channel is 60°, the oil phase inlet, the water phase inlet and the droplet outlet are on the same straight line, and the channel height is 50~100μm (the two oil phase channels and the droplet transport channel are similar to a Y-shaped structure).

[0038] Step S5: Dissolve the norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, photoinitiator 2959, and bergenin in phosphate buffered solution (PBS) at mass concentrations of 10–20 mg / mL, 50–100 mg / mL, 5–10 mg / mL, and 3–4 × 10⁻⁶ mg / mL, respectively. -4A solution of 50-100 mg / mL TWEEN20 is introduced as the aqueous phase through inlet 1 at a flow rate of 75-200 μL / h. The thiol-modified gelatin obtained in step S2 is dissolved in PBS solution and introduced as the aqueous phase through inlet 2. Mineral oil containing SPAN80 (CAS No. 8020-83-5) is introduced as the oil phase through inlet 2 at a flow rate of 400-1000 μL / h, with a SPAN80 mass fraction of 10%-20%. After the liquid is introduced, the aqueous and oil phases meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out through the droplet transport channels from the droplet outlet. The resulting microgel prepolymer microspheres are exposed to UV light for 3-5 min and then sequentially passed through mineral oil and PBS solution containing TWEEN20 (TWEEN20-83-5). The mixture was washed with PBS solution at a volume fraction of 0.5-1% to obtain a microgel loaded with bergenin (as a cartilage repair layer).

[0039] The norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, and photoinitiator 2959 were dissolved in PBS solution at concentrations of 10–20 mg / mL, 50–100 mg / mL, and 5–10 mg / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 1 at a flow rate of 75–200 μL / h. The thiol-modified gelatin and bone morphogenetic protein obtained in step S2 were dissolved in PBS solution at concentrations of 50–100 mg / mL and 80–120 ng / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 2. Mineral oil containing SPAN80 was introduced as the oil phase through the oil phase inlet at a flow rate of 400–1000 μL / h. μL / h; After the liquid is introduced, the aqueous phase and the oil phase meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out from the droplet outlet through the droplet transport channels. The obtained microgel prepolymer microspheres are exposed to ultraviolet light for 3 min to 5 min, and then washed sequentially with mineral oil, PBS solution containing TWEEN 20, and PBS solution to obtain microgels carrying bone morphogenetic proteins (as a bone repair layer).

[0040] Step S6: The microgels loaded with bergenin and bone morphogenetic proteins obtained in step S5 are stacked according to the preset cartilage damage area. They are then soaked in an aqueous solution of thiol-modified gelatin obtained in step S2 (the mass fraction of thiol-modified gelatin is 50~100 mg / mL, containing 5~10 mg / mL of photoinitiator 2959). After removing the excess liquid, the microgels are exposed to ultraviolet light for 15~30 s to obtain a "Janus" zoned drug delivery porous gel scaffold, which is used to fill the bone joint surface defect area and reshape the bone-cartilage interface.

[0041] In a preferred embodiment of the present invention, in step S1, the molecular weight of hyaluronic acid is 9~

[0042] 12 w.

[0043] In a preferred embodiment of the present invention, in step S2, the gelatin is pigskin gelatin or fish skin gelatin, and the dialysis bag material in both steps S2 and S1 is cellulose with a molecular weight cutoff of 8000.

[0044] 15000 Da.

[0045] In a preferred embodiment of the present invention, in step S3, the cyclodextrin is β-cyclodextrin, the dialysis belt is made of cellulose, and the molecular weight cutoff is 300~500 Da.

[0046] In a preferred embodiment of the present invention, in step S4, the etching gas pressure of the plasma etching is 6 mTorr, the etching temperature is 10°C, the etching power is 50 W for RF and 100 W for ICP, the etching time is 2-3 min, and the etching depth is 100-150 μm. The etching gas used is a single gas or a multi-component mixture such as oxygen, trifluoromethane / sulfur hexafluoride, or trifluoromethane / argon.

[0047] In a preferred embodiment of the present invention, in step S5, the bergenin and bone morphogenetic protein used can be replaced with other biological functional factors.

[0048] In a preferred embodiment of the present invention, in step S6, the microgel assembly process can also be carried out by using a 3D printer to finely print tissue-like structures.

[0049] A partitioned drug delivery porous gel scaffold prepared according to a preparation method.

[0050] Application of a zoned drug delivery porous gel scaffold in the preparation of drugs for treating osteoarthritis injuries.

[0051] Example 1: A method for preparing a partitioned drug delivery porous gel scaffold according to an embodiment of the present invention includes the following steps:

[0052] Step S1: Preparation of norbornene-modified hyaluronic acid: Dissolve 2 g of hyaluronic acid in 400 mL of deionized water. After complete dissolution, add 17.5 g of adipic acid dihydrazide and 4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Adjust the pH to 4.75 with 1 mol / L HCl aqueous solution throughout the process. After reacting for 12 h, pour the solution into a dialysis bag and dialyze for 4 days. Freeze-dry the dialysis solution to obtain adipic acid dihydrazide-modified hyaluronic acid. 2 g of adipic acid dihydrazide-modified hyaluronic acid was dissolved in 400 mL of deionized water. 4.5 g of cis-5-norbornene-endo-2,3-dicarboxylic anhydride was dissolved in 100 mL of dimethylformamide. This solution was added dropwise to the adipic acid dihydrazide-modified hyaluronic acid solution. The pH of the solution was adjusted to 7 with 1 mol / L NaOH aqueous solution throughout the process. After reacting for 12 h, the solution was poured into a dialysis bag and dialyzed for 4 days. The dialysis solution was then freeze-dried to obtain norbornene-modified hyaluronic acid, which was stored at -80 ℃.

[0053] Step S2: Preparation of thiol-modified gelatin: Dissolve 2 g of dimethyl 3,3-dithiodipropionate and 2 mL of hydrazine hydrate in 25 mL of methanol, react for 12 h, filter the suspension, and wash the resulting white solid thoroughly with methanol and deionized water for later use. Dissolve 2 g of gelatin in 200 mL of deionized water (50 ℃), add 0.5 g of the above white solid and 0.3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, react for 2 h, and then adjust the pH of the solution to 7 with 1 mol / L NaOH aqueous solution to terminate the reaction. Add 2.5 g of dithiothreitol to the above solution, react for 24 h, and then adjust the pH of the solution to 8.5 with 1 mol / L NaOH aqueous solution, and then adjust the pH of the solution to 3.5 with 1 mol / L HCl aqueous solution. The above reaction solution was poured into a dialysis bag and dialyzed in hydrochloric acid solution with pH=4.75 for 2 days, followed by dialysis in deionized water for 2 days. The dialysis solution was then freeze-dried to obtain mercapto-modified gelatin, which was stored at -80 °C.

[0054] Step S3: Preparation of double-bond modified cyclodextrin: Dissolve 10 g of cyclodextrin in 150 mL of dimethylformamide, add 3.5 mL of triethylamine and 2.5 mL of acryloyl chloride dropwise to the solution, react for 12 h, filter to remove solids, pour the resulting liquid into a dialysis bag and dialysis for 4 days, freeze dry to obtain double-bond modified cyclodextrin, and store at -80 ℃.

[0055] Step S4: Fabrication of droplet microfluidic chip: Place the obtained "Y"-shaped PDMS channel and the glass slide used as the bonding substrate in a plasma cleaner. Clean the PDMS channel surface and the glass slide with the surface facing upwards for 5 minutes. Immediately after removal, attach the PDMS channel surface to the cleaned surface of the silicon wafer substrate and slowly expel the air bubbles (because some air bubbles will remain in the middle during the bonding process, so it is necessary to slowly squeeze them out). Place the chip on a 120°C heating stage to remove the moisture between the channel and the silicon wafer substrate, so that the channel bonding is more complete.

[0056] Step S5: Dissolve the norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, photoinitiator 2959, and bergenin in PBS solution at concentrations of 20 mg / mL, 50 mg / mL, 5 mg / mL, and 3.2 × 10⁻⁶ mg / mL, respectively. -4 A solution of 50 mg / mL of thiol-modified gelatin obtained in step S2 was introduced into the aqueous phase inlet 1 of the droplet microfluidic chip at a flow rate of 100 μL / h. This solution was also introduced into the aqueous phase inlet 2 of the droplet microfluidic chip as the aqueous phase. Mineral oil containing SPAN80 was introduced into the oil phase inlet at a flow rate of 600 μL / h, with a SPAN80 mass fraction of 20%. After the liquid was introduced, the aqueous and oil phases met at the intersection of the channels, resulting in microemulsification. The aqueous phase was cut by the oil phase to form micron-sized droplets, which flowed out through the droplet transport channels from the droplet outlet. The resulting microgel prepolymer microspheres were exposed to UV light for 5 min and then washed sequentially with mineral oil, a PBS solution containing TWEEN 20, and another PBS solution to obtain a microgel loaded with bergenin (as a cartilage repair layer).

[0057] The norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, and the photoinitiator 2959 were dissolved in PBS solution at mass concentrations of 20 mg / mL, 50 mg / mL, and 5 mg / mL, respectively. These solutions were introduced into the aqueous phase of the droplet microfluidic chip through inlet 1 at a flow rate of 100 μL / h. The thiol-modified gelatin and bone morphogenetic protein obtained in step S2 were dissolved in PBS solution at concentrations of 50 mg / mL and 100 ng / mL, respectively. These solutions were introduced into the aqueous phase of the droplet microfluidic chip through inlet 2. Mineral oil containing SPAN80 was introduced as the oil phase through the oil phase inlet at a flow rate of 600 μL / h. After the liquid was introduced, the aqueous phase and the oil phase met at the intersection of the channels and underwent microemulsification. The aqueous phase was cut by the oil phase to form micron-sized droplets, which flowed out through the droplet transport channels from the droplet outlet. The obtained microgel prepolymer microspheres were exposed to ultraviolet light for 5 min and then washed sequentially with mineral oil, PBS solution containing TWEEN 20, and PBS solution to obtain microgels carrying bone morphogenetic proteins (as a bone repair layer).

[0058] Step S6: The microgels loaded with bergenin and bone morphogenetic proteins obtained in step S5 are stacked according to the preset cartilage damage area. They are then soaked in an aqueous solution of thiol-modified gelatin obtained in step S2 (the mass fraction of thiol-modified gelatin is 50 mg / mL, containing 5 mg / mL of photoinitiator 2959). After removing excess liquid, the microgels are exposed to ultraviolet light for 30 seconds to obtain a "Janus" zoned drug delivery porous gel scaffold, which is used to fill the bone joint surface defect area and reshape the bone-cartilage interface.

[0059] See Figure 1 (a) As can be seen, the droplet microfluidic chip is formed by bonding a layer of PDMS with a porous structure to a glass slide. It consists of three parts: an aqueous inlet 1 / 2 and aqueous transport channels, an oil inlet and oil transport channels, and a droplet outlet and droplet transport channels (scale bar: 1 cm); see also Figure 1 (b) It can be seen that at the intersection of the aqueous phase transport channel, the oil phase transport channel, and the droplet output channel, there is a rectangular channel with a length of 600 μm and a width of 300 μm (scale bar: 100 μm); see also Figure 1 (c) It can be seen that the polymerized microgel has strong overall uniformity (scale bar: 200 μm).

[0060] See Figure 3 (a) It can be seen that the synthesized microgels can be extruded by squeezing with a 2 ml syringe, and still retain their bulk structure after extrusion without collapsing (scale bar: 1 cm); see also Figure 3 (b) It can be seen that microgels can be 3D printed as needed, such as microtubes and microrod structures (scale bar: 2 mm); see also Figure 3(c) It can be seen that the synthesized partitioned drug delivery porous gel scaffold can be squeezed out by a 1 ml syringe needle, which facilitates drug delivery (scale bar: 2 cm).

[0061] See Figure 4 and Figure 5 Compared with the control group, the partitioned drug delivery porous gel scaffold synthesized in this invention has a better effect on promoting joint injury repair.

[0062] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a porous gel scaffold for zoned drug delivery, characterized in that, Includes the following steps: Step S1: Preparation of norbornene-modified hyaluronic acid: Dissolve 1-3 g of hyaluronic acid in deionized water. After complete dissolution, add 15-20 g of adipic acid dihydrazide and 3-5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Maintain the pH at 4.5-5 during the reaction. After 8-12 h of reaction, pour the solution into a dialysis bag and dialysis for 3-5 days. Freeze-dry the dialysis solution to obtain adipic acid dihydrazide-modified hyaluronic acid. Alternatively, dissolve 1-3 g of adipic acid dihydrazide-modified hyaluronic acid in deionized water. Dissolve 4-5 g of cis-5-norbornene-endo-2,3-dicarboxylic anhydride in 80-120 mL of dimethylformamide and add it dropwise to the adipic acid dihydrazide-modified hyaluronic acid solution. Maintain the pH at 6.5-7.5 during the reaction. After 8-12 h of reaction, pour the solution into a dialysis bag and dialysis for 3-5 days. d. After freeze-drying the dialysis solution, norbornene-modified hyaluronic acid was obtained and stored at -90 to -70 °C. Step S2: Preparation of thiol-modified gelatin: Dissolve 1-2 g of dimethyl 3,3-dithiodipropionate and 1-2 mL of hydrazine hydrate in 20-30 mL of methanol, react for 8-12 h, filter the suspension, and wash the resulting white solid with methanol and deionized water for later use; dissolve 1-2 g of gelatin in deionized water, add 0.3-1 g of the white solid and 0.1-1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, react for 1-3 h, adjust the pH of the solution to 7, and terminate the reaction; add 2-3 g of dithiothreitol to the solution, react for 20-30 h, adjust the pH of the solution to 8.5, and then adjust the pH of the solution to 3-4; pour the reaction solution into a dialysis bag, dialyze in hydrochloric acid solution with pH=4.5-5 for 1-3 days, and then dialyze in deionized water for 1-2 days. d. The dialysis solution was freeze-dried to obtain mercapto-modified gelatin, which was stored at -90 to -70 °C. Step S3: Preparation of double-bond modified cyclodextrin: Dissolve 8-12 g of cyclodextrin in 100-200 mL of dimethylformamide, add 3-4 mL of triethylamine and 2-3 mL of acryloyl chloride dropwise to the solution, react for 8-12 h, filter to remove solids, pour the resulting liquid into a dialysis bag and dialysis for 3-5 days, freeze dry to obtain double-bond modified cyclodextrin, and store at -90 to -70 ℃. Step S4: Prepare a droplet microfluidic chip; the droplet microfluidic chip includes an aqueous phase inlet 1, an aqueous phase inlet 2, an oil phase inlet, and a droplet outlet; Step S5: Dissolve the norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, photoinitiator 2959, and bergenin in PBS solution at mass concentrations of 10–20 mg / mL, 50–100 mg / mL, 5–10 mg / mL, and 3–4 × 10⁻⁶ mg / mL, respectively. -4 A solution of 50-100 mg / mL of thiol-modified gelatin obtained in step S2 is introduced into the aqueous phase through inlet 1 at a flow rate of 75-200 μL / h. This solution is then used as the aqueous phase through inlet 2. Mineral oil containing SPAN80 is introduced into the oil phase through inlet 400-1000 μL / h. After the liquid is introduced, the aqueous and oil phases meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out through the droplet transport channels from the droplet outlet. The resulting microgel prepolymer microspheres are exposed to ultraviolet light for 3-5 min, and then washed sequentially with mineral oil, a PBS solution containing TWEEN 20, and another PBS solution to obtain a microgel loaded with bergenin. The norbornene-modified hyaluronic acid obtained in step S1, the double-bond-modified cyclodextrin obtained in step S3, and photoinitiator 2959 were dissolved in PBS solution at concentrations of 10–20 mg / mL, 50–100 mg / mL, and 5–10 mg / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 1 at a flow rate of 75–200 μL / h. The thiol-modified gelatin and bone morphogenetic protein obtained in step S2 were dissolved in PBS solution at concentrations of 50–100 mg / mL and 80–120 ng / mL, respectively, and introduced as the aqueous phase through aqueous phase inlet 2. Mineral oil containing SPAN80 was introduced as the oil phase through the oil phase inlet at a flow rate of 400–1000 μL / h. μL / h; After the liquid is introduced, the aqueous phase and the oil phase meet at the intersection of the channels and undergo microemulsification. The aqueous phase is cut by the oil phase to form micron-sized droplets, which flow out from the droplet outlet through the droplet transport channels. The obtained microgel prepolymer microspheres are exposed to ultraviolet light for 3 min to 5 min, and then washed sequentially with mineral oil, PBS solution containing TWEEN 20, and PBS solution to obtain microgels carrying bone morphogenetic proteins. Step S6: The microgels loaded with bergenin and bone morphogenetic proteins obtained in step S5 are stacked according to the preset cartilage injury area. After being soaked in the thiol-modified gelatin aqueous solution obtained in step S2, excess liquid is removed. After a second UV exposure for 15-30 s, a zoned drug delivery porous gel scaffold is obtained.

2. The method for preparing a zoned drug delivery porous gel scaffold according to claim 1, characterized in that, In step S1, the molecular weight of hyaluronic acid is 9~12 w.

3. The method for preparing a zoned drug delivery porous gel scaffold according to claim 1, characterized in that, In step S2, the gelatin is pigskin gelatin or fish skin gelatin. The dialysis bags in both steps S2 and S1 are made of cellulose with a molecular weight cutoff of 8000~15000 Da.

4. The method for preparing a zoned drug delivery porous gel scaffold according to claim 1, characterized in that, In step S3, the cyclodextrin is β-cyclodextrin, the dialysis bag is made of cellulose, and the molecular weight cutoff is 300~500 Da.

5. The method for preparing a zoned drug delivery porous gel scaffold according to claim 1, characterized in that, In step S5, the mass fraction of SPAN80 in the mineral oil is 10%~20%.

6. The method for preparing a zoned drug delivery porous gel scaffold according to claim 1, characterized in that, In step S6, the mass concentration of thiol-modified gelatin is 50-100 mg / mL, and the aqueous solution of thiol-modified gelatin contains 5-10 mg / mL of photoinitiator 2959.

7. A partitioned drug delivery porous gel scaffold prepared by the preparation method according to any one of claims 1-6.

8. The use of a zoned drug delivery porous gel scaffold according to claim 7 in the preparation of a drug for treating osteoarthritis lesions.

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