CeSNs nano-enzyme preparation method and application of loaded butylphthalide in treatment of radioactive jawbone necrosis

Abstract

The invention provides a CeSNs nano-enzyme preparation method and application of loaded butylphthalide in treatment of radioactive jaw necrosis. Dendritic mesoporous silica nanoparticles (DMSN) are used as a template, cerium oxide doped silicate nano-enzyme (CeSNs) is prepared through a hydrothermal method, and the prepared nano-enzyme has antioxidant enzyme activity. Then, the nano-enzyme is loaded with NBP to obtain NBP-coated CeSNs, and the release of NBP and silicon ions in the NBP-coated CeSNs can improve angiogenesis and osteogenesis of the jaw bone. The nano-enzyme prepared by the research has great application prospect and value in the field of radioactive jawbone necrosis treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomedicine, and specifically relates to a method for preparing CeSNs nanozymes and application of CeSNs nanozymes loaded with butylphthalide in treating radioactive jaw osteonecrosis. Background Art

[0002] Radiotherapy-induced osteonecrosis of the jaw is a common complication of radiotherapy for patients with head and neck cancer. High doses of radiation not only kill tumor tissue, but also damage surrounding healthy tissue, leading to bone necrosis, chronic infection, and impaired healing. This disease can cause severe pain, uncontrolled infection, and dysfunction of the oral and maxillofacial system, which seriously affects the patient's quality of life. Currently, treatments for osteonecrosis of the jaw include antibiotics, surgical debridement, and hyperbaric oxygen therapy (HBO). However, these interventions have significant limitations. Antibiotics are generally unable to effectively penetrate poorly vascularized bones, while surgery carries the risk of exacerbating bone damage and further compromising structural integrity. Although HBO can promote healing by improving oxygenation of affected tissues, its effectiveness is limited and its effectiveness varies, often producing inconsistent results. These issues highlight the importance of finding new treatments for osteonecrosis of the jaw.

[0003] A key factor in radiation-induced injury is the overproduction of reactive oxygen species (ROS). ROS cause oxidative stress, promote inflammation and disrupt the delicate balance between bone formation and resorption. This imbalance accelerates tissue degradation and increases susceptibility to infection, complicating healing.

[0004] Nanozymes can mimic a series of antioxidant enzyme reactions to remove ROS, regulate the oxidative stress balance of biological tissues, and reduce inflammation. Nanozymes-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.

[0005] N-butylphthalide (NBP) was originally developed as a neuroprotectant and has now become a candidate drug for the treatment of oxidative stress and inflammation in various tissues. The potent anti-inflammatory properties of NBP can enhance mitochondrial efficiency, promote vascularization, and help restore angiogenesis in damaged tissues. In addition, silicon ions have a strong ability to stimulate osteoblast activity, which can enhance bone regeneration and support the formation of new bone. However, NBP alone stays in the lesion area for a long time, making it difficult for the drug to achieve a sustained therapeutic effect. Therefore, it is necessary to load NBP through nanocarriers to achieve the sustainability of NBP's therapeutic effect. However, pure NBP nanocarriers can only load drugs and do not have the ability to simulate antioxidant enzymes. Therefore, it is necessary to provide a nanozyme that can not only serve as a carrier of NBP, but also simulate the activity of antioxidant enzymes to achieve reactive oxygen scavenging, and further promote the treatment of radioactive jaw necrosis.

[0006] Here, we used dendritic mesoporous silica nanoparticles (DMSN) as templates to prepare cerium oxide-doped silicate nanozymes (CeSNs) by a hydrothermal method to mimic the antioxidant enzyme activities of SOD and CAT. Subsequently, NBP was loaded to obtain NBP@CeSNs. The release of NBP and silicon ions in NBP@CeSNs can improve angiogenesis and osteogenesis of the jaw. Based on this, the nanozymes prepared in this study have great application prospects and value in the treatment of radionecrosis of the jaw. Summary of the invention

[0007] In view of the shortcomings of the prior art, the first purpose of the present invention is to develop a CeSNs nanozyme that can simulate SOD and CAT reactions, achieve ROS clearance, and provide a technology for treating radionecrosis of the jaw.

[0008] The first object of the present invention is to provide a method for preparing CeSNs nanozymes, the method comprising the following steps:

[0009] S1. Dissolve DMSN in deionized water, dissolve polyvinyl pyrrolidone in deionized water, and mix the two solutions at room temperature to obtain a mixed solution; dissolve NH4Cl and Ce(NO3)3·6H2O in the mixed solution at room temperature, add 28% NH3·H2O solution, and react at 180°C for 24 hours to obtain a crude CeSNs product;

[0010] S2, washing the crude CeSNs obtained in S1 by centrifugation with deionized water and ethanol;

[0011] S3. After vacuum drying, CeSNs nanozymes are obtained.

[0012] Furthermore, the DMSN described in S1 is prepared by the following method:

[0013] Hexadecyltrimethylammonium p-toluenesulfonate, triethylamine and 1-butyl-3-methylimidazole trifluoromethanesulfonate are mixed in deionized water, stirred at 80°C for 1 hour, ethyl orthosilicate is added to the mixture, stirred at 80°C for 2 hours, centrifuged, and the precipitate is washed and purified with deionized water and ethanol respectively to obtain a crude DMSN product; hexadecyltrimethylammonium p-toluenesulfonate is removed from the prepared crude DMSN product by a template extraction method, an HCl ethanol solution is added, the suspension is ultrasonically treated for 2 hours, and then stirred at 70°C for 24 hours, the washing process is repeated, and DMSN is vacuum dried;

[0014] Preferably, the concentration of crude DMSN in HCl ethanol solution is in the range of 0.1-3 mg / mL;

[0015] Preferably, the HCl ethanol solution is obtained by mixing 12 mol / L HCl and ethanol in a volume ratio of 3:20;

[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 mass ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethylamine and 1-butyl-3-methylimidazolium trifluoromethanesulfonate is 96:105:10.

[0018] Furthermore, in S1:

[0019] The concentration of DMSN in deionized water was 0.5-5 mg / mL;

[0020] The concentration range of polyvinylpyrrolidone in deionized water is 1-10 mg / mL;

[0021] The mass ratio of DMSN to polyvinyl pyrrolidone is 1-10:1-10.

[0022] The mass ratio of DMSN to NH4Cl is 10-100:53-1060.

[0023] The mass ratio of NH4Cl to Ce(NO3)3·6H2O is 50-1000:43-430

[0024] Furthermore, in S2, the centrifugation is performed at 10,000 to 12,000 rpm for 10 to 30 min.

[0025] Furthermore, in S3, the vacuum drying is carried out at a temperature of 35 to 50° C. and for a time of 24 to 72 hours.

[0026] The second object of the present invention is to provide a CeSNs nanozyme, wherein the CeSNs nanozyme is prepared by the aforementioned preparation method;

[0027] Preferably, the components of the CeSNs nanozyme include cerium oxide and silicate;

[0028] Preferably, the CeSNs nanozyme has a spherical morphology and a particle size of 100 nm.

[0029] The third object of the present invention is to provide the use of the aforementioned CeSNs nanozyme in the preparation of a drug delivery system loaded with butylphthalide.

[0030] Furthermore, the CeSNs nanozyme loaded with butylphthalide can increase the release rate of silicon ions and butylphthalide under acidic conditions.

[0031] The fourth object of the present invention is to provide the use of the aforementioned CeSNs nanozyme or the drug delivery system loaded with butylphthalide as described in claim 8 in the preparation of a drug for treating radionecrosis of the jaw.

[0032] Beneficial effects of the present invention:

[0033] (1) The CeSNs nanozyme prepared by the present invention has a simple preparation method and is easy to synthesize.

[0034] (2) The CeSNs nanozymes prepared in the present invention have cascade antioxidant activity and can convert ·O2 - Converted into H2O2, and then decomposed into O2 using CAT-like activity.

[0035] (3) CeSNs can load NBP and have the function of pH-responsive NBP release.

[0036] (4) NBP@CeSNs have the pH-responsive function of releasing silicon ions, thereby promoting osteogenesis.

[0037] (5) NBP@CeSNs has the effect of promoting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a transmission electron micrograph of the CeSNs nanozyme prepared in Example 1 of the present invention; the figure shows that the nanozyme has a uniform particle size and good dispersibility, indicating that the prepared material has a nanoscale size.

[0039] Figure 2 This is the X-ray photoelectron spectrum of the CeSNs nanozyme prepared in Example 1 of the present invention. Signal peaks of Ce, Si and O elements can be seen, which proves that the components of the CeSNs nanozyme are cerium oxide and silicate.

[0040] Figure 3 The CeSNs 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.

[0041] Figure 4 This is the situation where the CeSNs nanozyme prepared in Example 1 of the present invention decomposes H2O2 to produce O2, indicating that the nanozyme has CAT-like activity.

[0042] Figure 5 The infrared spectra of CeSNs and NBP@CeSNs prepared in Examples 1 and 5 of the present invention are shown in Figure 1. Compared with the infrared spectra of CeSNs, NBP@CeSNs has a higher infrared spectrum at 1760 cm -1 The newly emerged absorption peak is the C=O absorption peak of NBP, indicating the successful combination of NBP and CeSNs.

[0043] Figure 6 This is the NBP release of NBP@CeSNs prepared in Example 5 of the present invention, indicating that the nanozyme has a pH-responsive NBP release function.

[0044] Figure 7 This is the situation of silicon ion release of NBP@CeSNs prepared in Example 5 of the present invention, indicating that the nanozyme has a pH-responsive silicon ion release function.

[0045] Figure 8 The NBP@CeSNs prepared in Example 5 of the present invention corrected the radioactive jaw necrosis in rats, indicating that the nanoparticles have the function of promoting bone regeneration in the tooth extraction wound of the rat tooth extraction model after the mandibular bone was irradiated with radiation.

[0046] Fig. 9 The NBP@CeSNs prepared in Example 5 of the present invention corrected the vascular inhibition in rat radioactive jaw necrosis, indicating that the nanoparticles have the function of promoting angiogenesis in the tooth extraction wound of the rat tooth extraction model after the mandibular bone was irradiated with radiation. DETAILED DESCRIPTION

[0047] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0048] Example 1

[0049] S1: Hexadecyltrimethylammonium p-toluenesulfonate (0.96 g), triethylamine (0.105 g) and 1-butyl-3-methylimidazolium trifluoromethanesulfonate (0.01 g) were first mixed in 50 mL of deionized water, and then the mixture was stirred at 80° C. for 1 hour.

[0050] Next, 7.8 mL of ethyl orthosilicate was quickly added to the mixture. After stirring at 80° C. for another 2 hours, the precipitate was purified by centrifugation and washed with deionized water and ethanol to obtain crude DMSN.

[0051] Then, the template extraction method was used to remove hexadecyltrimethylammonium p-toluenesulfonate from the prepared crude DMSN.

[0052] After adding HCl ethanol solution (mixture of 15 mL HCl (12 mol / L) and 100 mL ethanol) to 0.1 g of the prepared DMSN, the suspension was ultrasonically treated for 2 h and then stirred at 70° C. for 24 h. This washing process was repeated three times.

[0053] Finally, DMSN was obtained by vacuum drying at 45°C for 48 hours.

[0054] S2: 0.05 g of DMSN was fully dissolved in 20 mL of deionized water at room temperature; at the same time, 0.05 g of polyvinyl pyrrolidone was fully dissolved in 10 mL of deionized water at room temperature; then, the two solutions were fully mixed at room temperature.

[0055] Afterwards, 530 mg NH4Cl and 173 mg Ce(NO3)3·6H2O were fully dissolved in the mixed solution at room temperature; then, 1 mL NH3·H2O (28%) was added to the mixed solution under magnetic stirring.

[0056] Finally, the obtained mixture was transferred into a high-pressure reactor with a polytetrafluoroethylene liner, heated to 180° C., and reacted for 24 hours.

[0057] S3: After cooling to room temperature, the product was obtained by centrifugation at 11000 rpm for 10 minutes, and then washed three times by centrifugation with deionized water and ethanol.

[0058] S4: Finally, vacuum drying was performed at 45 °C for 48 h to obtain CeSNs.

[0059] Example 2

[0060] S1: Hexadecyltrimethylammonium p-toluenesulfonate (0.96 g), triethylamine (0.105 g) and 1-butyl-3-methylimidazolium trifluoromethanesulfonate (0.01 g) were first mixed in 50 mL of deionized water, and then the mixture was stirred at 80° C. for 1 hour.

[0061] Next, 7.8 mL of ethyl orthosilicate was quickly added to the mixture. After stirring at 80° C. for another 2 hours, the precipitate was purified by centrifugation and washed with deionized water and ethanol to obtain crude DMSN.

[0062] Then, the template extraction method was used to remove hexadecyltrimethylammonium p-toluenesulfonate from the prepared crude DMSN.

[0063] After adding HCl ethanol solution to 0.1 g of the prepared DMSN, the suspension was ultrasonically treated for 2 h and then stirred at 70 °C for 24 h. This washing process was repeated three times.

[0064] Finally, DMSN was obtained by vacuum drying at 40°C for 60 hours.

[0065] S2: 0.08 g of DMSN was fully dissolved in 20 mL of deionized water at room temperature; at the same time, 0.08 g of polyvinyl pyrrolidone was fully dissolved in 10 mL of deionized water at room temperature; then, the two solutions were fully mixed at room temperature.

[0066] Afterwards, 795 mg NH4Cl and 347 mg Ce(NO3)3·6H2O were fully dissolved in the mixed solution at room temperature; then, 1 mL NH3·H2O (28%) was added to the mixed solution under magnetic stirring.

[0067] Finally, the obtained mixture was transferred into a high-pressure reactor with a polytetrafluoroethylene liner, heated to 180° C., and reacted for 24 hours.

[0068] S3: After cooling to room temperature, the product was obtained by centrifugation at 11500 rpm for 8 minutes, and then washed three times by centrifugation with deionized water and ethanol.

[0069] S4: Finally, the CeSNs were obtained by vacuum drying at 40 °C for 60 h.

[0070] Example 3

[0071] The morphology of CeSNs prepared in Example 1 was analyzed by transmission electron microscopy. Figure 1 This is its transmission electron microscope image, which shows that the nanozyme has uniform particle size and good dispersion, indicating that the prepared material has nanoscale size.

[0072] Example 4

[0073] The elemental composition of CeSNs prepared in Example 1 was analyzed by X-ray photoelectron spectroscopy. Figure 2 Signal peaks of Ce, Si and O elements can be seen, which proves that the components of CeSNs nanozymes are cerium oxide and silicate.

[0074] Example 5

[0075] The CeSNs prepared in Example 1 were tested for their SOD-like activity using a SOD kit (Nanjing Jiancheng Bioengineering Institute, total superoxide dismutase (T-SOD) test kit (hydroxylamine method)) according to the kit instructions. Figure 3 As shown, it has good SOD-like activity. When the CeSNs concentration is 0.2 mg / mL, ·O2- The removal efficiency was 66%.

[0076] Example 6

[0077] The CeSNs prepared in Example 1 were mixed with H2O2 (8 mg CeSNs dissolved in 16 mL 200 mM H2O2), and the O2 release was detected by a dissolved oxygen meter. The CAT-like activity was detected. The experimental results are as follows: Figure 4 As shown, O2 can be generated immediately, indicating that CeSNs have good CAT-like activity.

[0078] Example 7

[0079] The CeSNs (10 mg) prepared in Example 1 and NBP (10 mg) were completely dissolved in 10 mL of deionized water at room temperature with continuous magnetic stirring for 24 h, and then the solution was centrifuged at 11000 rpm for 15 min, and the precipitate was washed with deionized water. Finally, NBP@CeSNs were obtained after vacuum drying.

[0080] Example 8

[0081] Appropriate amounts of CeSNs and NBP@CeSNs prepared in Example 1 and Example 7 were taken for KBr tablet pressing, and infrared absorption was tested using an infrared spectrometer.

[0082] The experimental results are as follows Figure 5 As shown in the figure, compared with the infrared spectrum of CeSNs, NBP@CeSNs has a -1 The newly emerged absorption peak is the C=O absorption peak of NBP, indicating the successful combination of NBP and CeSNs.

[0083] Example 9

[0084] 2 mg of NBP@CeSNs prepared in Example 7 was placed in a test tube, and then 1.5 mL of PBS (pH 6.6 or 7.4) was added to disperse it evenly, and then shaken at 37° C. The above dispersion was centrifuged at different time points, 250 μL of supernatant was taken out from the solution, the NBP content of the supernatant was determined, and then 250 μL of fresh PBS was added to the test tube.

[0085] The experimental results are as follows Figure 6 As shown, the release of NBP has a pH-responsive function, and the release rate is faster under acidic conditions, and about 60% of NBP can be released in 24 hours.

[0086] Example 10

[0087] 1 mg of NBP@CeSNs in Example 7 was placed in a test tube, and then 10 mL of PBS (pH 6.6 or 7.4) was added to disperse the NBP@CeSNs. Centrifugation was performed at 37°C at different time points, and 1 mL of supernatant was taken from the solution. The release of silicon ions in the supernatant was determined by ICP-OES, and 1 mL of fresh PBS was added to the NBP@CeSNs dispersion.

[0088] The experimental results are as follows Figure 7 As shown, the release of silicon ions has a pH response function, and the release rate is faster under acidic conditions. About 59% of Si can be released in 120 hours.

[0089] Embodiment 11

[0090] Twenty healthy 6-week-old adult male Sprague-Dawley rats were selected and randomly divided into a control group, a 40 Gy irradiation group, a 40 Gy irradiation + CeSNs group, and a 40 Gy irradiation + NBP@CeSNs group, with 5 rats in each group. The left mandible of the rats in the 40 Gy irradiation group, the 40 Gy irradiation + CeSNs group, and the 40 Gy irradiation + NBP@CeSNs group were irradiated using a small animal irradiator, with an irradiation dose of 8 Gy per day for 5 consecutive days, and a total dose of 40 Gy. During irradiation, the distance between the irradiation source and the rat skin was ensured to be 10 mm 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. CeSNs or NBP@CeSNs were locally injected into the tooth extraction wound, once 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.

[0091] like Figure 8 As shown in the data, both CeSNs and NBP@CeSNs can correct osteonecrosis of the jaw in rats and promote bone regeneration in the tooth extraction wound of a rat tooth extraction model after mandibular irradiation. NBP@CeSNs has a better effect due to the addition of NBP.

[0092] Example 12

[0093] The rat mandibular specimen in Example 11 was fixed, decalcified, and paraffin-sectioned and stained.

[0094] like Fig. 9 As shown in the results, CeSNs and NBP@CeSNs can promote the expression of VEGF in the tooth extraction wound of the rat tooth extraction model after mandibular irradiation, indicating that they have the function of promoting angiogenesis.

[0095] 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 CeSNs nanozyme, characterized in that: The method comprises the following steps: S1. Dissolve DMSN in deionized water, dissolve polyvinyl pyrrolidone in deionized water, and mix the two solutions at room temperature to obtain a mixed solution; dissolve NH4Cl and Ce(NO3)3·6H2O in the mixed solution at room temperature, add 28% NH3·H2O solution, and react at 180°C for 24 hours to obtain a crude CeSNs product; S2, washing the crude CeSNs obtained in S1 by centrifugation with deionized water and ethanol; S3. After vacuum drying, CeSNs nanozymes are obtained.

2. The preparation method according to claim 1, characterized in that: The DMSN described in S1 is prepared by the following method: hexadecyltrimethylammonium p-toluenesulfonate, triethylamine and 1-butyl-3-methylimidazole trifluoromethanesulfonate are mixed in deionized water, stirred at 80°C for 1 hour, ethyl orthosilicate is added to the mixture, stirred at 80°C for 2 hours, centrifuged, and the precipitate is washed and purified with deionized water and ethanol respectively to obtain a crude DMSN product; hexadecyltrimethylammonium p-toluenesulfonate is removed from the prepared crude DMSN product by a template extraction method, an HCl ethanol solution is added, the suspension is ultrasonically treated for 2 hours, and then stirred at 70°C for 24 hours, the washing process is repeated, and the DMSN is vacuum dried to obtain the DMSN; Preferably, the concentration of crude DMSN in HCl ethanol solution is in the range of 0.1-3 mg / mL; Preferably, the HCl ethanol solution is obtained by mixing 12 mol / L HCl and ethanol in a volume ratio of 3:20; 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 mass ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethylamine and 1-butyl-3-methylimidazolium trifluoromethanesulfonate is 96:105:

10.

4. The preparation method according to claim 1, characterized in that: In S1: The concentration of DMSN in deionized water was 0.5-5 mg / mL; The concentration range of polyvinylpyrrolidone in deionized water is 1-10 mg / mL; The mass ratio of DMSN to polyvinyl pyrrolidone is 1-10:1-10. The mass ratio of DMSN to NH4Cl is 10-100:53-1060. The mass ratio of NH4Cl to Ce(NO3)3·6H2O is 50-1000:43-430.

5. The preparation method according to claim 1, characterized in that: In S2, the centrifugation is carried out at 10000-12000 rpm for 10-30 min.

6. The preparation method according to claim 1, characterized in that: In S3, the vacuum drying is carried out at a temperature of 35 to 50°C and for a time of 24 to 72 hours.

7. CeSNs nanozyme, characterized in that The CeSNs nanozyme is prepared by the preparation method according to any one of claims 1 to 6; Preferably, the components of the CeSNs nanozyme include cerium oxide and silicate; Preferably, the CeSNs nanozyme has a spherical morphology and a particle size of 100 nm.

8. Use of the CeSNs 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 CeSNs nanozyme loaded with butylphthalide can increase the release rate of silicon ions and butylphthalide under acidic conditions.

10. Use of the CeSNs nanozyme according to claim 7 or the drug delivery system loaded with butylphthalide according to claim 8 in the preparation of a drug for treating radioactive osteonecrosis of the jaw.

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