Nanometer hydrogel loaded with dihydroartemisinin and preparation method thereof

Abstract

The invention relates to the technical field of medical treatment, and particularly discloses dihydroartemisinin-loaded nano-hydrogel, which is prepared from the following components: 5 to 20 percent of gelatin methacryloyl, 5 to 15 percent of nano-hydroxyapatite and 0.5 to 2 percent of dihydroartemisinin, according to the invention, through the constructed Ge MA-nHA-DHA functionalized hydrogel system, the local concentration and bioavailability of dihydroartemisinin are remarkably improved, and the repair and regeneration of bone tissues are promoted. Research results show that the system can effectively inhibit inflammatory response, promote proliferation and differentiation of osteoblasts, improve the structure and function of bone tissues, slow down further collapse of femoral heads and finally improve the living quality of patients. In addition, the biological material shows good biocompatibility and appropriate drug release characteristics when applied in vivo, and a novel effective means is provided for treatment of steroid-induced femoral head necrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a nano hydrogel loaded with dihydroartemisinin and a preparation method thereof. BACKGROUND

[0002] Hormone-induced femoral head necrosis is a kind of avascular necrosis of bone tissue caused by long-term use of hormone drugs, which mainly affects the structure and function of the femoral head. The existing treatment methods include conservative treatment and surgical treatment, but these methods often cannot effectively prevent the progression of the disease. In recent years, researchers have begun to explore new drug carrier systems to improve the local concentration and bioavailability of drugs. Dihydroartemisinin, as a drug with anti-inflammatory and bone formation promoting effects, has gradually attracted attention. Studies have shown that dihydroartemisinin can improve bone tissue repair by inhibiting inflammatory response and promoting the proliferation and differentiation of osteoblasts;

[0003] At present, the treatment methods for hormone-induced femoral head necrosis mainly focus on drug treatment and surgical intervention. In terms of drug treatment, commonly used drugs include non-steroidal anti-inflammatory drugs, bone protectants, etc., but the efficacy of these drugs is limited and may be accompanied by side effects. Surgical treatment includes femoral head replacement, etc., which can improve the quality of life of patients, but the surgical risk and recovery time are long. With the development of nanotechnology, nanocarrier systems have been gradually applied in the field of drug delivery, which can achieve targeted release and sustained release of drugs, and improve the therapeutic effect. Nanogel, as a new type of drug carrier, has good biocompatibility and adjustable release characteristics, and is suitable for local treatment of bone tissue.

[0004] Although the existing treatment methods can alleviate symptoms to some extent, there are still many shortcomings. First, the effect of traditional drug treatment is often not ideal, and it is difficult to achieve the ideal local drug concentration, resulting in insignificant treatment effect. Second, although surgical treatment can solve the structural problem, the postoperative recovery period is long, and there is a risk of complications. In addition, the existing drug carrier system still needs to be improved in terms of biocompatibility and drug release control. Therefore, there is an urgent need for a new treatment method that can effectively provide mechanical support while having anti-inflammatory and bone formation promoting functions to slow down the further collapse of the femoral head and improve the prognosis of patients. SUMMARY

[0005] The purpose of the present application is to provide a nano hydrogel loaded with dihydroartemisinin and a preparation method thereof to solve the problems raised in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] A nano hydrogel loaded with dihydroartemisinin and a preparation method thereof, comprising the following components:

[0008] Gelatin methacrylate 5-20%, nano-hydroxyapatite 5-15%, dihydroartemisinin 0.5-2% as therapeutic drugs.

[0009] Preferably, the particle size of the nano-hydroxyapatite is 50-100 nm, uniformly dispersed in the gelatin methacrylate matrix.

[0010] Preferably, the hydrogel has a porous fibrous or sheet-like microstructure, with a porosity of 60-85%.

[0011] A preparation method of the nano-hydrogel according to any one of the above, comprising the following steps:

[0012] (a) Dissolve gelatin methacrylate in phosphate buffer to a final concentration of 5-20% (w / v);

[0013] (b) Add nano-hydroxyapatite and ultrasonically disperse to a final concentration of 5-15% (w / v);

[0014] (c) Add dihydroartemisinin solution and mix evenly to a final concentration of 0.5-2% (w / v);

[0015] (d) Inject the mixed solution into a mold and perform photo-crosslinking solidification by irradiating with 405 nm ultraviolet light for 30-60 seconds.

[0016] Preferably, the optimal ratio is gelatin methacrylate 10%, nano-hydroxyapatite 10%, and dihydroartemisinin 1%.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] Through the constructed GelMA-nHA-DHA functionalized hydrogel system, the local concentration and bioavailability of dihydroartemisinin are significantly improved, and the repair and regeneration of bone tissue are promoted. The research results show that the system can effectively inhibit inflammatory response, promote the proliferation and differentiation of osteoblasts, improve the structure and function of bone tissue, slow down the further collapse of the femoral head, and ultimately improve the quality of life of patients. In addition, the application of biomaterials in vivo shows good biocompatibility and suitable drug release characteristics, providing a new and effective means for the treatment of hormone-induced femoral head necrosis. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the preparation flowchart of the present application;

[0020] Figure 2 is the microstructure schematic diagram of the hydrogel of the present application;

[0021] Figure 3Figure for cell migration and osteogenic differentiation experiment of the present application;

[0022] Figure 4 Figure for TUNEL apoptosis detection of the present application;

[0023] Figure 5 Figure for animal model surgery implantation of the present application;

[0024] Figure 6 Figure for Micro-CT bone morphological quantitative analysis of the present application;

[0025] Figure 7 Figure for histological staining of the present application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] Embodiment one:

[0028] Please refer to Figures 1-2 A nano-hydrogel loaded with dihydroartemisinin, comprising the following components:

[0029] Gelatin methacrylate 10%, nano-hydroxyapatite 10%, dihydroartemisinin 1%.

[0030] A preparation method of a nano-hydrogel, comprising the following steps:

[0031] Preparation of gelatin methacrylate (GelMA) prepolymer solution: dissolve GelMA in PBS buffer, final concentration 10% (w / v);

[0032] Preparation of nano-hydroxyapatite (nHA) suspension: uniformly disperse nHA in PBS buffer, final concentration 10% (w / v);

[0033] Preparation of dihydroartemisinin (DHA) solution: dissolve DHA in phosphate buffer (PBS), final concentration 1% (w / v);

[0034] Mix the three components in proportion: take 10% GelMA solution as the matrix, add 10% nHA suspension, vortex to uniformly disperse the nanoparticles, add 1% DHA solution, gently stir to avoid air bubbles, and form a homogeneous mixture (GelMA-nHA-DHA);

[0035] The mixed solution is injected into a mold (such as a cylindrical or disc shape), irradiated under 405 nm ultraviolet light for 30-60 seconds to trigger the photo-crosslinking reaction of GelMA, forming a solid hydrogel, and the solidified hydrogel is removed and washed with sterile PBS for 3 times to remove unreacted monomers.

[0036] Control group setting: synchronously prepare pure GelMA (10%) and GelMA-nHA (10% GelMA + 10% nHA) samples;

[0037] Scanning electron microscope (SEM) observation: freeze-dry the hydrogel sample, and after gold spraying treatment, observe the surface microstructure by SEM, as shown in Figure 2 The surface of the material of GelMA 10% is relatively smooth, with some smaller pores; the surface of the material of GelMA 10%-10% nHA has some granular substances; and the surface structure of the material of GelMA 10%-10% nHA-1% DHA is the most complex, with obvious fibrous or flaky substances.

[0038] Example Two:

[0039] As shown in Figure 3 , bone marrow mesenchymal stem cells (BMSCs) of rats with hormone-induced femoral head necrosis model were extracted, and the materials were co-cultured to evaluate the migration, osteogenic differentiation and apoptosis of BMSCs;

[0040] Cell extraction and culture: the rats were sacrificed, the femur and tibia were separated under sterile conditions, the bone marrow cavity was flushed with α-MEM medium, the flushing liquid was collected, the cells were resuspended after centrifugation (1000 rpm, 5 min), and the cells were inoculated in culture bottles using α-MEM medium containing 10% FBS and 1% double-antibiotic (penicillin / streptomycin) and cultured in a 37°C, 5% CO2 incubator, with medium change every 3 days, and the 3rd-5th passage cells were used for experiments;

[0041] Divided into experimental group: BMSCs+GelMA-nHA-DHA hydrogel; control group: BMSCs+GelMA-nHA hydrogel; blank group (without material treatment);

[0042] The sterilized hydrogel was cut into thin slices and placed at the bottom of a 24-well plate, 5×10 4 BMSCs were inoculated in each well, and complete culture medium was added;

[0043] Transwell vertical migration experiment: the hydrogel was placed in the lower chamber of the Transwell (24-well plate lower chamber), 2×10 4 BMSCs were inoculated in the upper chamber, and after 24 h of culture, the cells were fixed with 4% paraformaldehyde, the un-migrated cells in the upper chamber were wiped off with a cotton swab, and 0.1% crystal violet was used for staining for 20 min, and the migrated cells were counted under a microscope, as shown in Figure 3 (A).

[0044] Scratch migration experiment: monolayer BMSCs cell layer was formed in 6-well plates, a straight line scratch was made with a 200 μL gun head, after PBS washing, culture medium containing different hydrogel extracts was added, and the scratch area was photographed at 0 h and 8 h as shown in Figure 3 (B);

[0045] Alizarin red staining (mineralization ability): after co-culture for 21 days, the cells were fixed with 4% paraformaldehyde for 30 min, 1% alizarin red solution was added for staining for 20 min, PBS was washed, and calcium nodules (red deposits) were observed under a microscope as shown in Figure 3 (C);

[0046] ALP staining (early osteogenic differentiation): after co-culture for 7 days, BCIP / NBT kit was used for staining, and ALP positive areas were observed under a microscope as shown in Figure 3 (D);

[0047] TUNEL staining: after co-culture for 48 h, the cells were fixed with 4% paraformaldehyde, and the TUNEL kit was used according to the operation: cell membrane permeation (0.1% Triton X-100, 10 min), TUNEL reaction mixture was added (37°C, dark incubation for 60 min), DAPI was used for staining cell nucleus, and apoptotic cells (green fluorescence) were observed under a fluorescence microscope as shown in Figure 4 ;

[0048] Example 3

[0049] As shown in Figure 5 , a rat SONFH model was constructed, the biomaterial was implanted into the femoral head, and the therapeutic effect in vivo was detected, including the formation of new bone trabeculae and the change in the number of bone lacunar cells;

[0050] SD rats with a mass of about 170±20 g were selected, model group SD rats were intraperitoneally injected with lipopolysaccharide twice, 20 μg / kg each time, with an interval of 24 h. The rats were injected with methylprednisolone sodium succinate (40 mg / kg, Pfizer, China) in the hip muscle, with an interval of 24 h, 3 times a week, and continuously injected for 4 weeks to construct a hormone-induced femoral head necrosis model;

[0051] After the model was successfully constructed, the animals were operated on, a hole with a diameter of 2 mm and a depth of 3 mm was made in the femur, and the material was extruded and cured using 405 nm ultraviolet light. After continuous feeding for 6 weeks, the animals were sacrificed and the tissues were taken;

[0052] As shown in Figure 6 , Micro-CT bone morphological analysis:

[0053] Quantitative indicators: bone volume fraction (BV / TV) is the percentage of trabecular bone volume to total volume; trabecular number (Tb.N) is the number of trabeculae per unit length; trabecular thickness (Tb.Th) is the average thickness of trabeculae; and trabecular separation (Tb.Sp) is the average width of the gap between trabeculae;

[0054] As shown in the experimental results, the BV / TV, Tb.N and Tb.Th of the GelMA-nHA-DHA group were significantly higher than those of the GelMA group, the GelMA-nHA group, and the Tb.Sp of the GelMA-nHA-DHA group was significantly lower than that of the GelMA group and the GelMA-nHA group.

[0055] As shown in the experimental results, the BV / TV, Tb.N and Tb.Th of the GelMA-nHA-DHA group were significantly higher than those of the GelMA group, the GelMA-nHA group, and the Tb.Sp of the GelMA-nHA-DHA group was significantly lower than that of the GelMA group and the GelMA-nHA group. Figure 7 As shown in the experimental results, the BV / TV, Tb.N and Tb.Th of the GelMA-nHA-DHA group were significantly higher than those of the GelMA group, the GelMA-nHA group, and the Tb.Sp of the GelMA-nHA-DHA group was significantly lower than that of the GelMA group and the GelMA-nHA group.

[0056] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A nano-hydrogel loaded with dihydroartemisinin, characterized in that, It comprises the following components: Gelatin methacrylate 5-20%, nano-hydroxyapatite 5-15%, dihydroartemisinin 0.5-2% as therapeutic drugs.

2. The nano-hydrogel loaded with dihydroartemisinin according to claim 1, characterized in that: The nano-hydroxyapatite has a particle size of 50-100 nm and is uniformly dispersed in the gelatin methacrylate matrix.

3. The nano-hydrogel loaded with dihydroartemisinin according to claim 1, characterized in that: The hydrogel has a porous fibrous or sheet-like microstructure, and the porosity is 60-85%.

4. A method of preparing the nanohydrogel according to any one of claims 1 to 3, characterized in that, It comprises the following steps: (a) Dissolve gelatin methacrylate in a phosphate buffer to a final concentration of 5-20% (w / v); (b) Add nano-hydroxyapatite and ultrasonically disperse it to a final concentration of 5-15% (w / v); (c) Add a dihydroartemisinin solution and mix well to a final concentration of 0.5-2% (w / v); (d) Inject the mixed solution into a mold and perform photo-crosslinking solidification by irradiating it with 405 nm ultraviolet light for 30-60 seconds.

5. The nano-hydrogel loaded with dihydroartemisinin according to claim 1, characterized in that: The most preferred ratio is gelatin methacrylate 10%, nano-hydroxyapatite 10%, and dihydroartemisinin 1%.

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