Preparation method of Mn and Ca double-site nano-enzyme and application of loaded butylphthalide
By developing the Mn and Ca two-site nanoenzymes, the problems of limited effect of ROS clearance therapy and single nanocarrier function in the prior art were solved, and the efficient treatment of ORNJ was achieved. Through the dual effects of ROS clearance and NBP sustained release, jaw regeneration and angiogenesis were promoted.
Patent Information
- Application Number
- CN202510195128.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art In the treatment of radioactive jaw osteomyelitis (ORNJ), the ROS clearance therapy has limited effect, and the traditional nanocarrier function is single, so it is impossible to achieve stable drug sustained release and ROS clearance at the same time.
A Mn and Ca two-site nanoenzyme was developed, prepared using mesoporous silica as a template. It has the dual enzyme simulation characteristics of SOD and CAT, which can clear ROS and achieve stable sustained release of the drug by loading butylphthalide (NBP).
It achieved efficient clearance of ROS and stable sustained release of NBP, promoted jaw regeneration and angiogenesis, and significantly improved the therapeutic effect of ORNJ.
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Figure CN119971075A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of nanobiotechnology, and specifically relates to a method for preparing a Mn and Ca dual-site nanozyme and an application of nanozyme loaded with butylphthalide in treating radiation-induced jaw osteomyelitis. Background Art
[0002] Osteoradionecrosis of the jaws (ORNJ) refers to a disease of prolonged jaw necrosis and soft tissue exposure in the radiation-damaged area after radiotherapy for head and neck tumors. It is accompanied by symptoms such as bone surface exposure, wound nonhealing, limited mouth opening, and pathological fractures. It is one of the most serious complications after radiotherapy for head and neck tumors. Epidemiological statistics show that the incidence of ORNJ in patients who receive head and neck radiotherapy is approximately 5%-15%. However, the etiology of ORNJ has not been clearly defined and treatment options are limited. A key factor in radiation-induced damage is the excessive production of reactive oxygen species (ROS). ROS can cause oxidative stress, promote inflammation, and disrupt the delicate balance between bone formation and bone resorption. This imbalance accelerates tissue degeneration and increases susceptibility to infection, complicating healing.
[0003] The causes of ORNJ are very complex, among which the replacement of jaw tissue in the irradiated area by fibrous tissue is one of the main possible factors. Several months after radiotherapy, the damaged tissue releases a large amount of reactive oxygen species, destroying the natural barrier of endothelial cells and activating the fibroblast activity of tissue cells; several years after radiotherapy, various cytokines, such as TNF-α, IL-1, IL-4, etc., are released in large quantities, prompting the continuous production of fibroblast phenotypes and engulfing surrounding tissues; 5-30 years after radiotherapy, fibroblast apoptosis disappears and forms fibrotic jaws, which eventually leads to ORNJ.
[0004] Nanozyme-induced superoxide dismutase (SOD) and catalase (CAT) enzyme cascade catalysis can eliminate superoxide anions (·O2 -) and hydrogen peroxide (H2O2). Cerium oxide nanozymes with dual enzyme mimetic properties of SOD and CAT can scavenge ROS. However, single ROS scavenging therapy has limited therapeutic effect on radionecrosis of the jaw, and the formation of new blood vessels and bone in the jaw microenvironment is crucial for the treatment of radionecrosis of the jaw. Inspired by enzyme-like catalysis, nanozymes with superoxide dismutase (SOD) and catalase (CAT) activities have been developed for the treatment of diseases related to ROS overexpression, but the catalytic efficiency of metal sites is often easily limited. Multi-metal elements can confer a large number of catalytic active sites and can enhance enzyme-like catalytic activity through multi-site interactions. Therefore, it is necessary to develop a new therapeutic material that can be used to treat ORNJ. N-butylphthalide (NBP) can expand blood vessels and promote jaw bone regeneration. NBP can be effectively loaded through nanocarriers for the treatment of ORNJ. However, traditional nanocarriers have a single function and only have the ability to release NBP slowly. The Mn and Ca dual-site nanozyme we developed can not only load NBP to achieve stable and sustained release of the drug, but also has the function of ROS scavenging. At the same time, the Si and Ca elements contained in the nanozyme can promote osteogenesis, which are all beneficial for the treatment of ORNJ. Summary of the invention
[0005] To solve the above technical problems in the prior art, the present invention develops a Mn, Ca dual-site nanozyme that can catalyze SOD-like and CAT-like reactions, achieve ROS removal, and provide a technology for treating ORNJ. Mn, Ca dual-site nanozyme is prepared using mesoporous silica as a template, and its main component elements Si and Ca can promote bone regeneration and further promote the treatment of ORNJ. The Mn, Ca dual-site nanozyme can also load butylphthalide (NBP) to promote jaw neovascularization. Based on this, the Mn, Ca dual-site nanozyme prepared in this study can have great application prospects and value in the field of ORNJ treatment after loading NBP.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing a Mn and Ca dual-site nanozyme comprises the following steps:
[0008] Dissolve mesoporous silica, CaCl2 and manganese acetate in water and continue stirring for 1 hour to obtain a mixed solution A;
[0009] Prepare 0.4mmol / mL NH4Cl aqueous solution, then add 28% ammonia water to mix to obtain mixed solution B;
[0010] Mixed solution B was added dropwise to mixed solution A, reacted at 200°C for 12 hours, cooled to room temperature, centrifuged, and the precipitate was washed with water and ethanol respectively, and vacuum dried to obtain the Mn and Ca dual-site nanozyme;
[0011] Preferably, the vacuum drying condition is vacuum drying at 35-50° C. for 24-72 hours.
[0012] Furthermore, the mesoporous silica is prepared by the following method:
[0013] Dissolve hexadecyltrimethylammonium p-toluenesulfonate in water, then add triethylamine and 1-butyl-3-methylimidazolium trifluoromethanesulfonate, stir the mixture at 80°C for 1 hour, add ethyl orthosilicate to the mixture, stir at 80°C for 2 hours, centrifuge, centrifuge with deionized water and ethanol, respectively, and dry to obtain a precipitate;
[0014] Washing the precipitate: Add the precipitate to a mixture of hydrochloric acid and ethanol, sonicate for 2 hours, stir at 70°C for 24 hours, and collect the precipitate by centrifugation;
[0015] The collected precipitate is washed with ethanol, centrifuged, and vacuum dried to obtain mesoporous silica;
[0016] Preferably, the vacuum drying is carried out at a temperature of 35 to 50° C. and for a time of 24 to 72 hours.
[0017] Furthermore, the concentration of hexadecyltrimethylammonium p-toluenesulfonate in water is 15-20 mg / mL, and the mass ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethylamine, and 1-butyl-3-methylimidazolium trifluoromethanesulfonate is 96:105:10.
[0018] Furthermore, the mass volume ratio of hexadecyltrimethylammonium p-toluenesulfonate to tetraethyl orthosilicate is 0.1-0.2 g / ml.
[0019] Furthermore, the mass volume ratio of the precipitate to the mixed solution of hydrochloric acid and ethanol is 1:1150, and the volume ratio of hydrochloric acid to ethanol in the mixed solution of hydrochloric acid and ethanol is 12 mol / L HCl and ethanol are mixed in a volume ratio of 3:20.
[0020] Furthermore, the concentration of the mesoporous silica in water is 0.002 g / mL, and the molar ratio of CaCl2 to manganese acetate is 1 to 30: 10 to 100. In a particular embodiment, the molar ratio of CaCl2 to manganese acetate is 1:1 to 1:5.
[0021] Furthermore, the volume ratio of the NH4Cl aqueous solution to 28% ammonia water is 100:1-5:1, and the volume ratio of the mixed solution A to B is 2.47:1-2.08:1.
[0022] The second object of the present invention is to provide a Mn, Ca dual-site nanozyme, which is prepared by the aforementioned preparation method.
[0023] The third object of the present invention is to provide the use of the aforementioned Mn, Ca dual-site nanozyme in the preparation of a drug delivery system loaded with butylphthalide.
[0024] Furthermore, the Mn and Ca dual-site nanozyme is loaded with butylphthalide and can release butylphthalide slowly.
[0025] The fourth object of the present invention is to provide the use of the aforementioned Mn, Ca dual-site nanozyme or the aforementioned butylphthalide-loaded drug delivery system in the preparation of a drug for treating radioactive osteonecrosis of the jaw.
[0026] Beneficial effects of the present invention:
[0027] (1) The Mn and Ca dual-site nanozyme prepared by the present invention has a simple preparation method and is easy to synthesize.
[0028] (2) The Mn and Ca dual-site nanozymes prepared by the present invention have cascade antioxidant activity and can convert ·O2 - Converted into H2O2, and then decomposed into O2 using CAT-like activity.
[0029] (3) Mn and Ca dual-site nanozymes can be loaded with NBP and achieve stable sustained release of drugs.
[0030] (4) Mn and Ca dual-site nanozymes loaded with NBP can treat ORNJ. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is a transmission electron microscopy image of the Mn and Ca dual-site nanozyme prepared in Example 1 of the present invention; the image shows that the nanozyme has a nanoscale size.
[0032] Figure 2 This is the X-ray photoelectron spectrum of the Mn, Ca dual-site nanozyme prepared in Example 1 of the present invention. Signal peaks of Mn, Ca, Si and O elements can be seen, which proves that the nanozyme is doped with Mn and Ca elements and has dual metal sites.
[0033] Figure 3 The Mn and Ca dual-site nanozyme prepared in Example 1 of the present invention removes O2 - The efficiency of the nanozyme was 0.1%, indicating that the nanozyme had SOD-like activity.
[0034] Figure 4 This is the situation where the Mn and Ca dual-site nanozyme prepared in Example 1 of the present invention decomposes H2O2 to produce O2, indicating that the nanozyme has CAT-like activity.
[0035] Figure 5The infrared spectra of the Mn, Ca dual-site nanozymes prepared in Examples 1 and 7 of the present invention and the Mn, Ca dual-site nanozymes loaded with NBP are shown in Figure 1. Compared with the infrared spectra of the Mn, Ca dual-site nanozymes, the Mn, Ca dual-site nanozymes loaded with NBP have an infrared spectrum at 2956 cm -1 The newly emerged absorption peak is the CH absorption peak of NBP, indicating that the Mn and Ca dual-site nanozyme was successfully loaded onto NBP.
[0036] Figure 6 This is the drug release situation of the Mn and Ca dual-site nanozyme loaded with NBP prepared in Example 7 of the present invention, indicating that NBP can be slowly released from the nanozyme.
[0037] Figure 7 The Mn and Ca dual-site nanozyme loaded with NBP prepared in Example 7 of the present invention corrected the radioactive jaw necrosis in rats, indicating that the nanozyme has the function of promoting bone regeneration in the tooth extraction wound of the rat tooth extraction model after the mandible was irradiated with radiation.
[0038] Figure 8 This is the case where the NBP-loaded Mn and Ca dual-site nanozyme prepared in Example 7 of the present invention promotes the growth of endothelial cells, indicating that the nanozyme has the function of promoting angiogenesis. DETAILED DESCRIPTION
[0039] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.
[0040] Example 1
[0041] Step 1: 0.96 g of hexadecyltrimethylammonium p-toluenesulfonate was dissolved in 50 mL of water, followed by the addition of 0.105 g of triethylamine and 0.01 g of 1-butyl-3-methylimidazolium trifluoromethanesulfonate, and the mixture was stirred at 80° C. for 1 hour.
[0042] Next, 7.8 mL of ethyl orthosilicate was added to the above solution, and the mixture was stirred at 80° C. for 2 hours, and then centrifuged to obtain a precipitate, which was then centrifuged with water and ethanol and dried to obtain a precipitate.
[0043] The above 0.1 g of precipitate was added to a mixture of 12 mol / L hydrochloric acid (15 mL) and ethanol (100 mL), ultrasonicated for 2 hours, stirred at 70°C for 24 hours, and centrifuged to collect the precipitate. The above steps were repeated three times, washed once with ethanol, centrifuged, and vacuum dried at 40°C for 48 hours to obtain mesoporous silica.
[0044] Step 2: Dissolve 0.05 g of mesoporous silica, 0.125 mmol of CaCl2 and 0.25 mmol of manganese acetate in 25 mL of water and continue stirring for 1 hour to obtain a mixed solution A.
[0045] 4 mmol of NH4Cl was dissolved in 10 mL of water, and then 0.4 mL of aqueous ammonia (28%) was added and mixed to obtain a mixed solution B.
[0046] Under stirring, 10.4 mL of mixed solution B was added dropwise to 25 mL of mixed solution A. After mixing completely, the mixture was transferred to a high-pressure reactor and reacted at 200 °C for 12 hours. After cooling to room temperature, the mixture was centrifuged, the precipitate was washed three times with water / ethanol, and vacuum dried at 40 °C for 48 hours to obtain the Mn, Ca dual-site nanozyme.
[0047] Example 2
[0048] Step 1: 0.96 g of hexadecyltrimethylammonium p-toluenesulfonate was dissolved in 50 mL of water, followed by the addition of 0.105 g of triethylamine and 0.01 g of 1-butyl-3-methylimidazolium trifluoromethanesulfonate, and the mixture was stirred at 80° C. for 1 hour.
[0049] Next, 7.8 mL of ethyl orthosilicate was added to the above solution, and the mixture was stirred at 80° C. for 2 hours, and then centrifuged to obtain a precipitate, which was then centrifuged with water and ethanol and dried to obtain a precipitate.
[0050] The above 0.1 g of precipitate was added to a mixture of 12 mol / L hydrochloric acid (15 mL) and ethanol (100 mL), ultrasonicated for 2 hours, stirred at 70°C for 24 hours, and centrifuged to collect the precipitate. The above steps were repeated three times, washed once with ethanol, centrifuged, and vacuum dried at 40°C for 48 hours to obtain mesoporous silica.
[0051] Step 2: Dissolve 0.05 g of mesoporous silica, 0.15 mmol of CaCl2 and 0.35 mmol of manganese acetate in 25 mL of water and continue stirring for 1 hour to obtain a mixed solution A.
[0052] 4 mmol of NH4Cl was dissolved in 10 mL of water, and then 0.6 mL of aqueous ammonia (28%) was added and mixed to obtain a mixed solution B.
[0053] Under stirring, 10.6 mL of mixed solution B was added dropwise to 25 mL of mixed solution A. After mixing was complete, the mixture was transferred to a high-pressure reactor and reacted at 200 °C for 12 hours. After cooling to room temperature, the mixture was centrifuged, the precipitate was washed three times with water / ethanol, and vacuum dried at 40 °C for 48 hours to obtain the Mn, Ca dual-site nanozyme.
[0054] Example 3
[0055] The morphology of the Mn and Ca dual-site nanozyme prepared in Example 1 was analyzed by transmission electron microscopy.
[0056] Figure 1 This is a transmission electron microscope image, which shows that the nanozyme has nanoscale size.
[0057] Example 4
[0058] The elemental composition of the Mn and Ca dual-site nanozyme prepared in Example 1 was analyzed by X-ray photoelectron spectroscopy.
[0059] like Figure 2 Signal peaks of Mn, Ca, Si and O elements can be seen, which proves that Mn and Ca are successfully doped into the nanozyme.
[0060] Example 5
[0061] The Mn and Ca dual-site nanozyme prepared in Example 1 was tested for its SOD-like activity using a SOD kit.
[0062] The experimental results are as follows Figure 3 As shown, it has good SOD-like activity. When the concentration of Mn and Ca dual-site nanozymes is 200 μg / mL, ·O2 - The removal efficiency is about 80%.
[0063] Example 6
[0064] The Mn and Ca dual-site nanozyme prepared in Example 1 was mixed with H2O2, and the O2 release was detected using a dissolved oxygen meter to detect its CAT-like activity.
[0065] The experimental results are as follows Figure 4 As shown, O2 can be generated quickly and the O2 concentration can reach over 30 mg / L in 6 minutes.
[0066] Example 7
[0067] Furthermore, the Mn, Ca dual-site nanozyme (1 mg) and NBP (3 mg) were dissolved and dispersed in 10 mL of water by magnetic stirring at room temperature, magnetically stirred in the dark for 1 day, and then the precipitate was washed three times by centrifugation with water. Finally, the Mn, Ca dual-site nanozyme loaded with NBP was obtained after vacuum drying.
[0068] Example 8
[0069] Appropriate amounts of the Mn, Ca dual-site nanozymes prepared in Example 1 and Example 7 and the Mn, Ca dual-site nanozymes loaded with NBP were pressed into KBr tablets, and infrared absorption was tested using an infrared spectrometer.
[0070] The experimental results are as follows Figure 5As shown in the figure, compared with the infrared spectrum of the Mn and Ca dual-site nanozyme, the Mn and Ca dual-site nanozyme loaded with NBP has a peak at 2956 cm -1 The newly emerged absorption peak is the CH absorption peak of NBP, indicating that the Mn and Ca dual-site nanozyme was successfully loaded onto NBP.
[0071] Example 9
[0072] The NBP-loaded Mn and Ca dual-site nanozyme in Example 7 was dispersed in 1.5 mL PBS and shaken at 37° C. The dispersion was centrifuged at different time points, 150 μL of supernatant was taken out from the solution, and the ultraviolet absorption of the supernatant was measured at 274 nm to evaluate the NBP release, and then 150 μL of fresh PBS was added to the test tube.
[0073] The experimental results are as follows Figure 6 As shown, NBP can be stably and slowly released from the nanozyme, and about 50% of NBP can be released within 72 hours.
[0074] Example 10
[0075] Twenty healthy 6-week-old adult male Sprague-Dawley rats were selected and randomly divided into a control group, a 40Gy irradiation group, a 40Gy irradiation + Mn, Ca dual-site nanozyme group, and a 40Gy irradiation + Mn, Ca dual-site nanozyme loaded with NBP group, with 5 rats in each group. The left mandible of the rats in the 40Gy irradiation group, the 40Gy irradiation + Mn, Ca dual-site nanozyme group, and the 40Gy irradiation + Mn, Ca dual-site nanozyme loaded with NBP group were irradiated using a small animal irradiator, with an irradiation dose of 8Gy per day for 5 consecutive days, and a total dose of 40Gy. During irradiation, ensure that the distance between the irradiation source and the rat skin is 10mm to ensure the accuracy of local irradiation. After the irradiation, rest for 10 days to ensure the stability of the irradiation effect. Then, teeth were extracted from the left mandibular area of rats in all groups, and 3 molars were extracted. Mn, Ca dual-site nanozymes or Mn, Ca dual-site nanozymes loaded with NBP were injected locally into the tooth extraction wound every 3 days for 21 days. After the experiment, the rats were euthanized and the left mandibular specimens were obtained for CT scanning and three-dimensional reconstruction.
[0076] The experimental results are as follows Figure 7 As shown, both the Mn, Ca dual-site nanozyme group and the Mn, Ca dual-site nanozyme group loaded with NBP can correct radioactive jaw necrosis in rats, and have the function of promoting bone regeneration in the tooth extraction wound of the rat tooth extraction model after the mandible was irradiated with radiation. The effect of the Mn, Ca dual-site nanozyme loaded with NBP is more obvious.
[0077] Embodiment 11
[0078] Endothelial cells were co-cultured with Mn, Ca dual-site nanozymes or Mn, Ca dual-site nanozymes loaded with NBP and subjected to AM / PI staining.
[0079] The experimental results are as follows Figure 8 As shown, it shows that the Mn, Ca dual-site nanozyme group and the Mn, Ca dual-site nanozyme group loaded with NBP both have good affinity for endothelial cells and have the function of promoting angiogenesis, and the effect of the Mn, Ca dual-site nanozyme loaded with NBP is more obvious.
[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing a Mn and Ca dual-site nanozyme, characterized in that: The following steps are involved: Dissolve mesoporous silica, CaCl2 and manganese acetate in water and continue stirring for 1 hour to obtain a mixed solution A; Prepare 0.4mmol / mL NH4Cl aqueous solution, then add 28% ammonia water to mix to obtain mixed solution B; Mixed solution B was added dropwise to mixed solution A, reacted at 200°C for 12 hours, cooled to room temperature, centrifuged, and the precipitate was washed with water and ethanol respectively, and vacuum dried to obtain the Mn and Ca dual-site nanozyme; Preferably, the vacuum drying condition is vacuum drying at 35-50° C. for 24-72 hours.
2. The preparation method according to claim 1, characterized in that: The mesoporous silica is prepared by the following method: dissolving hexadecyltrimethylammonium p-toluenesulfonate in water, then adding triethylamine and 1-butyl-3-methylimidazole trifluoromethanesulfonate, stirring the mixture at 80° C. for 1 hour, adding ethyl orthosilicate to the mixture, stirring at 80° C. for 2 hours, centrifuging, centrifuging with deionized water and ethanol, respectively, and drying to obtain a precipitate; Washing the precipitate: Add the precipitate to a mixture of hydrochloric acid and ethanol, sonicate for 2 hours, stir at 70°C for 24 hours, and collect the precipitate by centrifugation; The collected precipitate is washed with ethanol, centrifuged, and vacuum dried to obtain mesoporous silica; Preferably, the mass volume ratio of hexadecyltrimethylammonium p-toluenesulfonate to ethyl orthosilicate is 0.1 to 0.2 g / ml; Preferably, the vacuum drying is carried out at a temperature of 35 to 50° C. and for a time of 24 to 72 hours.
3. The preparation method according to claim 2, characterized in that: The concentration of the hexadecyltrimethylammonium p-toluenesulfonate in water is 15-20 mg / mL, and the mass ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethylamine, and 1-butyl-3-methylimidazole trifluoromethanesulfonate is 96:105:
10.
4. The preparation method according to claim 2, characterized in that: The mass volume ratio of the precipitate to the mixed solution of hydrochloric acid and ethanol is 1:1150, and the mixed solution of hydrochloric acid and ethanol is 12 mol / L HCl and ethanol mixed in a volume ratio of 3:
20.
5. The preparation method according to claim 1, characterized in that: The concentration of the mesoporous silica in water is 0.002 g / mL, the molar ratio of CaCl2 to manganese acetate is 1-30:10-100, and preferably, the molar ratio of CaCl2 to manganese acetate is 1:1-1:
5.
6. The preparation method according to claim 1, characterized in that: The volume ratio of the NH4Cl aqueous solution to 28% ammonia water is 100:1-5:1, and the volume ratio of the mixed solution A to B is 2.47:1-2.08:
1.
7. Mn, Ca dual-site nanozyme, characterized in that: The Mn, Ca dual-site nanozyme is prepared by the preparation method described in any one of claims 1 to 6.
8. Use of the Mn and Ca dual-site nanozyme according to claim 7 in the preparation of a drug delivery system loaded with butylphthalide.
9. The use according to claim 8, characterized in that: The Mn and Ca dual-site nanozyme is loaded with butylphthalide and can release butylphthalide slowly.
10. Use of the Mn, Ca dual-site nanozyme according to claim 7 or the butylphthalide-loaded drug delivery system according to claim 9 in the preparation of a drug for treating radioactive osteonecrosis of the jaw.