Preparation method of novel pH-sensitive DHA (at) ZIF-8NPs and application of novel pH-sensitive DHA (at) ZIF-8NPs in preparation of medicine for treating sarcopenia-osteoporosis

Abstract

The invention discloses a preparation method of novel pH-sensitive DHA (at) ZIF-8NPs and application of the novel pH-sensitive DHA (at) ZIF-8NPs in preparation of a medicine for treating sarcopenia-osteoporosis, and relates to the technical field of research and development of the medicine for treating sarcopenia-osteoporosis. The preparation method comprises the following steps: step 1, dissolving 75 mg of zinc nitrate hexahydrate in 2.5 ml of deionized water to obtain a solution I, dissolving 165 mg of 2-methylimidazole in 4.5 ml of methanol to obtain a solution II, and dissolving 10 mg of DHA in 0.5 ml of DMF to obtain a solution III; step 2, adding the solution I into a mixture of the solution II and the solution III while stirring at room temperature, and obtaining a mixed solution after the synthetic solution becomes turbid rapidly for 3 minutes; step 3, centrifuging the mixed solution for 10 minutes at the rotating speed of 12000 rpm, so as to obtain DHA (at) ZIF-8NPs; 4, washing DHA (at) ZIF-8NPs with methanol for three times to completely remove unreacted reagents, and collecting all supernate for measuring the packaging efficiency and the drug loading content of DHA; and step 5, finally, performing freeze drying on the DHA (at) ZIF-8NPs, and performing low-temperature storage.

Description

Technical Field

[0001] The present invention relates to the technical field of sarcopenia-osteoporosis drug research and development, and specifically to a preparation method of novel pH-sensitive DHA@ZIF-8NPs and an application thereof in the preparation of a drug for treating sarcopenia-osteoporosis. Background Art

[0002] Dihydroartemisinin (DHA) is an important drug developed in my country based on artemisinin. It has significant therapeutic effects in the treatment of malaria. Due to the poor stability and low water solubility of DHA, its bioavailability is affected and drug resistance is easily generated. Therefore, it is of great significance to improve the water solubility and stability of DHA. In the treatment of bone-related diseases, nanobiomaterials have broad prospects and great potential. Current research has applied them to diseases such as bone defects, osteoporosis, osteoarthritis, primary and metastatic bone cancer. Nanomaterials can selectively target bone tissue and cells to deliver drugs, which can solve the problems related to poor stability and low water solubility of DHA. However, the current selection and preparation of materials are still in the exploratory stage, and research in this field needs to be further deepened.

[0003] Zeolite-imidazolate frameworks (ZIFs) are a common MOFs material with a wide range of applications. Among them, ZIF-8 has excellent stability, and after reaching a weakly acidic microenvironment, the material can gradually degrade and release Zn2+ and the drugs it carries. Sarcopenia-osteoporosis is often related to increased inflammatory response in the body. During the inflammatory response, the body will secrete a large amount of lactic acid as a metabolite. The accumulation of lactic acid will lead to local tissue acidification, thereby changing the inflammatory microenvironment. Studies have shown that the damaged bone microenvironment is mostly acidic. According to the acid-sensitive properties of ZIF-8, the increased inflammatory response causes the lesion site in the body to become acidic, which can accurately and quickly release the drugs encapsulated in the nanocarrier to achieve precise disease treatment.

[0004] The muscle and skeletal systems are closely connected. In recent years, the number of patients with sarcopenia-osteoporosis has gradually increased, but there are few effective treatments. Based on the interaction between muscle and bone lesions, it is particularly important to explore their common therapeutic targets. Studies in recent years have shown that DHA has potential in promoting bone formation and inhibiting bone resorption. Studies have found that DHA can dose-dependently inhibit RANKL-induced osteoclastogenesis and bone resorption. In in vivo experimental studies, DHA can reverse bone loss caused by ovariectomy. However, in current experimental studies, few researchers have paid attention to the effect of DHA on muscle lesions, and there is a lack of research on the application of DHA in the treatment of sarcopenia-osteoporosis.

[0005] Therefore, we proposed a novel pH-sensitive DHA@ZIF-8NPs preparation method and its application in the preparation of drugs for the treatment of sarcopenia-osteoporosis.

[0006] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method of novel pH-sensitive DHA@ZIF-8NPs and its application in the preparation of drugs for the treatment of sarcopenia-osteoporosis, to improve the shortcomings of dihydroartemisinin such as poor water solubility and low bioavailability that limit clinical application, and to explore and improve the application of dihydroartemisinin in the preparation of drugs for the treatment of sarcopenia-osteoporosis.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing novel pH-sensitive DHA@ZIF-8NPs, comprising the following steps:

[0009] Step 1: Dissolve 75 mg of zinc nitrate hexahydrate in 2.5 ml of deionized water to obtain solution 1, dissolve 165 mg of 2-methylimidazole in 4.5 ml of methanol to obtain solution 2, and dissolve 10 mg of DHA in 0.5 ml of dimethylformamide (DMF) to obtain solution 3;

[0010] Step 2: Add solution 1 to the mixture of solution 2 and solution 3 under stirring at room temperature. The resultant solution quickly becomes turbid. After 3 minutes, a mixed solution is obtained.

[0011] Step 3: Centrifuge the mixed solution at 12000 rpm for 10 minutes to obtain DHA@ZIF-8NPs;

[0012] Step 4: Wash DHA@ZIF-8NPs three times with methanol to completely remove the unreacted reagents, and collect all the supernatants for measuring the encapsulation efficiency and drug loading content of DHA;

[0013] Step 5: Finally, DHA@ZIF-8NPs were freeze-dried and stored at low temperature.

[0014] Preferably, in step 5, the DHA@ZIF-8NPs are stored at minus 20 degrees Celsius.

[0015] The present invention also provides DHA@ZIF-8 prepared by the preparation method of the novel pH-sensitive DHA@ZIF-8NPs as described above.

[0016] The present invention also provides the use of the above-mentioned DHA@ZIF-8 in the preparation of a drug for treating sarcopenia-osteoporosis.

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

[0018] (1) Under the preparation process of the present invention, ZIF-8 successfully encapsulates DHA, and the prepared DHA@ZIF-8 has improved water solubility compared with DHA. The results of transmission electron microscopy experiments, X-ray diffraction experiments, nanoparticle size potentiometer detection experiments, and infrared spectroscopy detection experiments show that the prepared DHA@ZIF-8 nanoparticles are uniform and stable, have a higher degree of crystallinity, and have a more stable dispersion system than ZIF-8. ZIF-8 is a good nanocarrier for DHA.

[0019] (2) In the in vitro cell experiment verification, the results of CCK-8 experiment, myoblast MHC staining, osteoclast TRAP staining and phalloidin staining experiments proved that the DHA@ZIF-8 prepared by the present invention is less cytotoxic to myoblasts and osteoclasts than DHA, and also has a certain enhancing effect in inhibiting osteoclast formation and promoting the formation of muscle cells.

[0020] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the results of transmission electron microscopy experiment (A, B), X-ray diffraction experiment (C), and nanoparticle size potential instrument detection experiment (D, E) of the present invention;

[0022] Figure 2 This is a schematic diagram of the infrared spectrum detection experimental results of the present invention;

[0023] Figure 3 This is a schematic diagram of the C2C12 cytotoxicity test results of the present invention;

[0024] Figure 4 This is a schematic diagram of the cytotoxicity test results of RAW264.7 cells of the present invention;

[0025] Figure 5 This is a schematic diagram of the osteoclast TRAP staining results of the present invention;

[0026] Figure 6 This is a schematic diagram of the results of osteoclast phalloidin staining of the present invention;

[0027] Figure 7 This is a schematic diagram of the myoblast MHC immunofluorescence results of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The preparation method of the novel pH-sensitive DHA@ZIF-8NPs comprises the following steps:

[0030] Step 1: Dissolve 75 mg of zinc nitrate hexahydrate in 2.5 ml of deionized water to obtain solution 1, dissolve 165 mg of 2-methylimidazole in 4.5 ml of methanol to obtain solution 2, and dissolve 10 mg of DHA in 0.5 ml of dimethylformamide (DMF) to obtain solution 3;

[0031] Step 2: Add solution 1 to the mixture of solution 2 and solution 3 under stirring at room temperature. The resultant solution quickly becomes turbid. After 3 minutes, a mixed solution is obtained.

[0032] Step 3: Centrifuge the mixed solution at 12000 rpm for 10 minutes to obtain DHA@ZIF-8NPs;

[0033] Step 4: Wash DHA@ZIF-8NPs three times with methanol to completely remove the unreacted reagents, and collect all the supernatants for measuring the encapsulation efficiency and drug loading content of DHA;

[0034] Step 5: Finally, DHA@ZIF-8NPs were freeze-dried and stored at low temperature.

[0035] The successfully prepared DHA@ZIF-8 was used to intervene in myoblasts and osteoclasts, and cell experiments were carried out to explore the toxic effects of DHA@ZIF-8 on normal myoblasts and osteoclasts, as well as its effects on the proliferation and differentiation abilities of damaged cells.

[0036] Transmission electron microscopy results Figure 1 As shown in Figures A and B, the particle size of the DHA@ZIF sample is about 50 nm, and the particle size of the blank ZIF sample is about 50 nm. The particle sizes of the two are uniform and both have good three-dimensional structures.

[0037] XRD results are as follows Figure 1As shown in Figure C, the upper figure is the DHA@ZIF sample, and the lower figure is the blank ZIF. The peaks of ZIF at 2θ=7.30, 10.35, 12.70, 14.80, 16.40 and 18.00 correspond to (110), (200), (211), (220), (310) and (222), respectively. The main diffraction peaks indicate that the sample ZIF has a high degree of crystallinity. The peaks of DHA@ZIF at 2θ=7.30, 10.35, 12.70, 14.80, 16.40 and 18.00 correspond to (110), (200), (211), (220), (310) and (222), respectively. The main diffraction peaks indicate that the sample DHA@ZIF has a high degree of crystallinity. And compared with the blank ZIF, the morphology has not changed.

[0038] The experimental results of nanoparticle size potential instrument are as follows Figure 1 As shown in Figures D and E, the average particle size of the DHA@ZIF sample prepared by the normal process is 197.9. The value of the blank ZIF prepared by the normal process is 107.1, indicating that the drug loading is successful.

[0039] The infrared spectrum detection experimental results are as follows Figure 2 As shown, the results indicate that ZIF-8 has a good DHA loading effect and the structure of the synthesized drug is stable.

[0040] In summary, the DHA@ZIF-8 nanoparticles prepared in the present invention are uniform and stable, have a higher degree of crystallinity, and have a more stable dispersion system than ZIF-8. ZIF-8 is a good nanocarrier for DHA.

[0041] CCK-8 test results Figure 3-Figure 4 As shown, compared with the control group, DHA@ZIF-8 at a concentration of 20μg / ml significantly inhibited the proliferation activity of C2C12, and at a concentration of 10μg / ml, it had a certain promoting effect on the proliferation activity of C2C12. Compared with the control group, DHA@ZIF-8 at a concentration of 1μg / ml and DHA at a concentration of 0.5-1μg / ml had an inhibitory effect on the proliferation activity of RAW264.7, DHA@ZIF-8 at a concentration of 0.25-0.5μg / ml had no cytotoxicity, and DHA at a concentration of 0.25 had no cytotoxicity. The difference was statistically significant.

[0042] RAW264.7 cells were induced to undergo osteoclastogenesis, and osteoclast TRAP staining was performed ( Figure 5 ) and phalloidin staining ( Figure 6 )The results showed that DHA@ZIF-8 drug can significantly inhibit the abnormal proliferation of osteoclasts induced by LPS.

[0043] Myoblast MHC immunofluorescence results Figure 7As shown, DHA and DHA@ZIF-8 drugs can significantly inhibit the damage of myocyte myotubes under LPS intervention.

[0044] In summary, the DHA@ZIF-8 prepared by the present invention has less cytotoxicity to myoblasts and osteoclasts than DHA, and also has a certain enhancing effect in inhibiting osteoclast formation and promoting the formation of myocytes.

[0045] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0046] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing novel pH-sensitive DHA@ZIF-8NPs, characterized in that: The steps include: Step 1: Dissolve 75 mg of zinc nitrate hexahydrate in 2.5 ml of deionized water to obtain solution 1, dissolve 165 mg of 2-methylimidazole in 4.5 ml of methanol to obtain solution 2, and dissolve 10 mg of DHA in 0.5 ml of DMF to obtain solution 3; Step 2: Add solution 1 to the mixture of solution 2 and solution 3 under stirring at room temperature. The resultant solution quickly becomes turbid. After 3 minutes, a mixed solution is obtained. Step 3: Centrifuge the mixed solution at 12000 rpm for 10 minutes to obtain DHA@ZIF-8NPs; Step 4: Wash DHA@ZIF-8NPs three times with methanol to completely remove the unreacted reagents, and collect all the supernatants for measuring the encapsulation efficiency and drug loading content of DHA; Step 5: Finally, DHA@ZIF-8NPs were freeze-dried and stored at low temperature.

2. The method for preparing the novel pH-sensitive DHA@ZIF-8NPs according to claim 1, characterized in that: In step 5, the DHA@ZIF-8NPs are stored at minus 20 degrees Celsius.

3. DHA@ZIF-8 prepared by the preparation method of the novel pH-sensitive DHA@ZIF-8NPs as described in any one of claims 1-2.

4. Use of DHA@ZIF-8 as described in claim 3 in the preparation of drugs for treating sarcopenia-osteoporosis.

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