A muscle administration type selenium nanoparticle preparation for spinal cord injury treatment

Abstract

The present application relates to the technical field of biological medicine, and disclose a kind of muscle administration type selenium nanoparticle preparation for spinal cord injury treatment, the selenium nanoparticle preparation is made from the following weight parts of raw materials: selenium nanoparticle 60-80 parts, cholera toxin B subunit 5-15 parts, dopamine hydrochloride 5-10 parts, curcumin 2-8 parts, and surfactant 1-5 parts;With selenium nanoparticle as delivery core, its surface is coated by polydopamine coating, improve the stability and biocompatibility of particle, reduce the non-specific clearance in body circulation;At the same time, the polydopamine coating provides active interface for functional modification, covalently connected cholera toxin B subunit on it can recognize and combine the specific receptor of surrounding nerve ending, selenium nanoparticle loaded with curcumin is delivered from muscle injection site to damaged spinal cord region, and the accuracy of treatment is enhanced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an intramuscularly administered selenium nanoparticle formulation for the treatment of spinal cord injury. Background Technology

[0002] Spinal cord injury occurs when external force acts directly or indirectly on the spinal cord, causing structural and functional changes that result in sensory, motor, and sphincter dysfunction below the level of injury, abnormal muscle tone, and pathological reflexes. Selenium is an essential trace element for the human body, playing important roles such as antioxidation, immune enhancement, anti-inflammation, and tumor inhibition. Daily intake of a certain amount of selenium can maintain normal bodily functions and help reduce the incidence of disease.

[0003] Currently, when using nanoparticle formulations to treat spinal cord injury, the complex local microenvironment after injury makes it difficult for conventional nanoparticles to efficiently accumulate in the damaged spinal cord area after being administered via intramuscular injection. This results in insufficient drug accumulation concentration at the target site and limited therapeutic efficacy.

[0004] Therefore, a selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury is proposed to address the above-mentioned problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an intramuscularly administered selenium nanoparticle formulation for the treatment of spinal cord injury, which solves the problem of insufficient drug accumulation concentration at the target site and limited therapeutic effect mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an intramuscular selenium nanoparticle formulation for the treatment of spinal cord injury, wherein the selenium nanoparticle formulation is made from the following raw materials in parts by weight: 60-80 parts selenium nanoparticles, 5-15 parts cholera toxin B subunit, 5-10 parts dopamine hydrochloride, 2-8 parts curcumin, and 1-5 parts surfactant.

[0007] Preferably, the selenium nanoparticles are prepared by the following method: 50-100 mg of selenocysteine ​​is added to 20-50 ml of water. Under magnetic stirring, the pH of the mixture is adjusted to 8.5-9.5 with a 0.1-1.0 M NaOH solution. The mixture is then continuously shaken and reacted at 50°C in the dark for 20-28 hours. After the reaction, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3000-5000 Da. Dialysis is performed in deionized water at 4-10°C for 12-24 hours, with the dialysis fluid replaced every 4-8 hours. After dialysis, the liquid in the dialysis bag is centrifuged at 4°C and 10000-15000 g / min for 15-30 minutes. The supernatant is discarded, and the resulting precipitate is the selenium nanoparticle. The precipitate is resuspended in a 0.01-0.5 M phosphate buffer solution with a pH of 7.2-7.6 to adjust its concentration to 5-10 mg. Se / ml, stored at 4℃ for later use, the resulting resuspended solution is the selenium nanoparticle stock suspension.

[0008] Preferably, the cholera toxin B subunit is covalently linked to the selenium nanoparticles. The specific linking method is as follows: a stock suspension of selenium nanoparticles is washed 1-3 times with 0.01-0.1M 2-(N-morpholine)ethanesulfonic acid buffer, and then mixed with 5-15 parts by weight of cholera toxin B subunit, 4-8 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 2-4 parts by weight of N-hydroxysuccinimide in ultrapure water, controlling the overall reaction system... The volume is 5-15 ml. The mixture is gently shaken and reacted at room temperature and in the dark for 1-3 hours. After the reaction is complete, the mixture is washed 2-4 times with ultrapure water at 4°C by centrifugation at 8000-12000 g / min for 10-20 minutes each time. Finally, the mixture is resuspended in phosphate buffer solution with a pH of 7.2-7.6 and a concentration of 0.01-0.2 M to obtain a selenium nanoparticle-cholera toxin B subunit complex suspension. This suspension is the selenium nanoparticle complex suspension.

[0009] Preferably, the concentration of dopamine hydrochloride added is 0.1-0.3 mg / ml, and it is combined with selenium nanoparticles in any of the following ways:

[0010] Dopamine hydrochloride was added to the selenium nanoparticle stock suspension, and the pH of the selenium nanoparticle stock suspension was adjusted to 8.5-9.5. The suspension was then incubated together at 25-37℃ for 2-6 hours to allow dopamine to self-polymerize on the surface of the selenium nanoparticles to form a polydopamine coating layer.

[0011] After the selenium nanoparticles were synthesized in a stock suspension, dopamine hydrochloride and the selenium nanoparticles were incubated together in an alkaline buffer solution with a pH of 8.0-9.0 at 25-37°C for 2-6 hours, so that dopamine self-polymerized on the surface of the selenium nanoparticles to form a polydopamine coating layer.

[0012] Preferably, the method for loading curcumin is as follows: curcumin is dissolved in anhydrous ethanol to prepare a curcumin ethanol solution with a concentration of 1-5 mg / ml. Under light-protected conditions, the curcumin ethanol solution is added dropwise to the obtained selenium nanoparticle complex suspension, and the final concentration of ethanol is controlled not to exceed 5% of the total volume of the solution. Then, under room temperature and light-protected conditions, the mixture is magnetically stirred at a speed of 100-300 rpm for 8-16 hours. After loading is completed, the mixture is centrifuged at 4℃ and 12000-15000 g / min for 20-30 minutes to remove unloaded free curcumin.

[0013] Preferably, after centrifugation to remove unloaded free curcumin, the process further includes a precipitation resuspension step: at 4°C, phosphate buffer is added to the selenium nanoparticle precipitate after removing free curcumin for resuspension, and the volume is adjusted to a final concentration of 1.0-5.0 mgSe / mL. The precipitate is then placed on a magnetic stirrer and stirred continuously at 100-300 rpm for 10-20 minutes to ensure that the precipitate is fully dispersed in the buffer solution. The mixture is then transferred to a sterile centrifuge tube and centrifuged again at 8000-10000 g / min for 5-10 minutes. The supernatant is discarded to remove any remaining undispersed particles. The resulting selenium nanoparticle resuspension is a uniformly dispersed intramuscularly administered selenium nanoparticle formulation.

[0014] Preferably, the phosphate buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration of 0.01-0.1M and a pH of 7.2-7.6, and the total sodium ion concentration is 0.02-0.2M.

[0015] Preferably, the surfactant is selected from one of polysorbate-80, Pluronic F-127, polyvinylpyrrolidone K30, and sodium dodecyl sulfate. The surfactant is added as follows: under room temperature and light-protected conditions, the surfactant is slowly added dropwise to the selenium nanoparticle suspension at a rate of 0.5-2.0 mL / min, and the mixture is continuously stirred at a speed of 200-600 rpm during the addition process. After the addition is completed, stirring is continued for 10-30 minutes to ensure that the surfactant is uniformly dispersed.

[0016] Preferably, the selenium nanoparticle formulation is adjusted to pH 7.2-7.6 with sterile phosphate buffer or physiological saline before use and sterilized by passing it through a 0.22μm sterile filter membrane.

[0017] Preferably, the selenium nanoparticle formulation is packaged in a vial or pre-filled syringe in unit dose form, with a unit dose volume of 0.5-2.0 mL, and is administered via intramuscular injection.

[0018] Compared with the prior art, the present invention provides an intramuscularly administered selenium nanoparticle formulation for the treatment of spinal cord injury, which has the following beneficial effects:

[0019] 1. In this invention, selenium nanoparticles are used as the delivery core, and their surface is coated with a polydopamine coating layer, which improves the stability and biocompatibility of the particles and reduces non-specific clearance in the systemic circulation. At the same time, the polydopamine coating layer provides an active interface for functional modification. The cholera toxin B subunit covalently linked on it can recognize and bind to specific receptors in peripheral nerve endings, delivering curcumin-loaded selenium nanoparticles from the intramuscular injection site to the damaged spinal cord area. This solves the problems of poor targeting and insufficient accumulation at the lesion site of conventional nano-preparations, and enhances the accuracy of treatment.

[0020] 2. In this invention, the functional component selenium nanoparticles are combined with the loaded drug curcumin to construct a synergistic therapeutic system. The selenium nanoparticles themselves possess antioxidant and anti-inflammatory activities, capable of neutralizing excessive harmful reactive oxygen species at the site of injury; the loaded curcumin exerts anti-inflammatory and neuroprotective effects. Both are co-delivered via a nanocarrier, producing a synergistic effect at the site of injury, thereby achieving synergistic intervention in oxidative stress and inflammatory responses after spinal cord injury, and improving the overall therapeutic effect.

[0021] 3. In this invention, the added surfactant prevents the aggregation of nanoparticles during storage and maintains their uniform dispersion at the nanoscale. The final formulation undergoes pH adjustment and aseptic filtration to form a physicochemically stable aseptic suspension that can be directly used for intramuscular injection, ensuring stability throughout the entire process from preparation and storage to drug administration, thus laying the foundation for the feasibility of clinical translation. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: An intramuscular selenium nanoparticle formulation for the treatment of spinal cord injury, the selenium nanoparticle formulation being made from the following raw materials in parts by weight: 60 parts selenium nanoparticles, 5 parts cholera toxin B subunit, 5 parts dopamine hydrochloride, 2 parts curcumin, and 1 part surfactant.

[0024] Selenium nanoparticles were prepared by the following method: 50 mg of selenocysteine ​​was added to 20 ml of water. Under magnetic stirring, the pH of the mixture was adjusted to 8.5 with 0.1 M NaOH solution. The mixture was then continuously shaken and reacted at 50 °C in the dark for 20 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4 °C for 12 hours. The dialysis solution was replaced every 4 hours. After dialysis, the liquid in the dialysis bag was centrifuged at 10000 g / min at 4 °C for 15 minutes. The supernatant was discarded, and the resulting precipitate was the selenium nanoparticles. The precipitate was resuspended in 0.01 M phosphate buffer solution with a pH of 7.2 to adjust the concentration to 5 mg Se / ml and stored at 4 °C for later use. The resulting resuspended solution was the selenium nanoparticle stock suspension.

[0025] The cholera toxin B subunit and selenium nanoparticles were covalently linked. The specific linking method was as follows: a stock suspension of selenium nanoparticles was washed once with 0.01M 2-(N-morpholine) ethanesulfonic acid buffer, and then mixed with 5 parts by weight of cholera toxin B subunit, 4 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 parts by weight of N-hydroxysuccinimide in ultrapure water. The total volume of the reaction system was controlled at 5 ml. The reaction was carried out with gentle shaking at room temperature and in the dark for 1 hour. After the reaction was completed, the mixture was washed twice with ultrapure water at 4°C by centrifugation at 8000 g / min for 10 minutes each time. Finally, the mixture was resuspended in 0.01M phosphate buffer at pH 7.2 to obtain the selenium nanoparticle-cholera toxin B subunit complex suspension.

[0026] The concentration of dopamine hydrochloride added was 0.1 mg / ml, and it was combined with selenium nanoparticles in the following manner:

[0027] Dopamine hydrochloride was added to the selenium nanoparticle stock suspension, and the pH of the selenium nanoparticle stock suspension was adjusted to 8.5. The suspension was then incubated together at 25°C for 2 hours to allow dopamine to self-polymerize on the surface of the selenium nanoparticles to form a polydopamine coating layer.

[0028] The loading method for curcumin was as follows: curcumin was dissolved in anhydrous ethanol to prepare a curcumin ethanol solution with a concentration of 1 mg / ml. Under light-protected conditions, the curcumin ethanol solution was added dropwise to the obtained selenium nanoparticle complex suspension, and the final concentration of ethanol was controlled not to exceed 5% of the total volume of the solution. Then, under room temperature and light-protected conditions, the mixture was magnetically stirred at 100 rpm for 8 hours. After loading was completed, the mixture was centrifuged at 4℃ and 12000 g / min for 20 minutes to remove unloaded free curcumin.

[0029] After centrifugation to remove unloaded free curcumin, the process includes a precipitation resuspension step: at 4°C, phosphate buffer is added to the selenium nanoparticle precipitate after free curcumin removal for resuspension, and the volume is adjusted to a final concentration of 1.0 mg Se / mL. The mixture is then placed on a magnetic stirrer and stirred continuously at 100 rpm for 10 minutes to ensure that the precipitate is fully dispersed in the buffer solution. The mixture is then transferred to a sterile centrifuge tube and centrifuged again at 8000 g / min for 5 minutes. The supernatant is discarded to remove any remaining undispersed particles. The resulting selenium nanoparticle resuspension is a uniformly dispersed intramuscular selenium nanoparticle formulation.

[0030] The phosphate buffer solution is a 0.01M, pH 7.2 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a total sodium ion concentration of 0.02M.

[0031] The surfactant was selected from polysorbate-80 and was added as follows: at room temperature and in the dark, the surfactant was slowly added dropwise to the selenium nanoparticle suspension at a rate of 0.5 mL / min, and the mixture was continuously stirred at a speed of 200 rpm during the addition process. After the addition was completed, the mixture was stirred for 10 minutes to ensure that the surfactant was evenly dispersed.

[0032] Before use, the selenium nanoparticle formulation was adjusted to pH 7.2 with sterile phosphate buffer and sterilized by passing it through a 0.22μm sterile filter membrane.

[0033] The selenium nanoparticle formulation is packaged in a vial in unit dose form, with a unit dose volume of 0.5 mL, and is administered via intramuscular injection.

[0034] Example 2: An intramuscular selenium nanoparticle formulation for the treatment of spinal cord injury, the selenium nanoparticle formulation being made from the following raw materials in parts by weight: 70 parts selenium nanoparticles, 10 parts cholera toxin B subunit, 8 parts dopamine hydrochloride, 5 parts curcumin, and 3 parts surfactant.

[0035] Selenium nanoparticles were prepared by the following method: 70 mg of selenocysteine ​​was added to 35 ml of water. Under magnetic stirring, the pH of the mixture was adjusted to 9.0 with 0.5 M NaOH solution. The mixture was then continuously shaken and reacted at 50 °C in the dark for 24 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 4500 Da and dialyzed in deionized water at 6 °C for 18 hours. The dialysis solution was replaced every 6 hours. After dialysis, the liquid in the dialysis bag was centrifuged at 12000 g / min at 4 °C for 20 minutes. The supernatant was discarded, and the resulting precipitate was the selenium nanoparticles. The precipitate was resuspended in 0.3 M phosphate buffer solution with a pH of 7.4 to adjust the concentration to 7 mg Se / ml and stored at 4 °C for later use. The resulting resuspended solution was the selenium nanoparticle stock suspension.

[0036] The cholera toxin B subunit and selenium nanoparticles were covalently linked. The specific linking method was as follows: a stock suspension of selenium nanoparticles was washed twice with 0.05M 2-(N-morpholine)ethanesulfonic acid buffer. Then, it was mixed with 10 parts by weight of cholera toxin B subunit, 6 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 3 parts by weight of N-hydroxysuccinimide in ultrapure water. The total volume of the reaction system was controlled at 10 ml. The reaction was carried out with gentle shaking at room temperature and in the dark for 2 hours. After the reaction was completed, the mixture was washed three times with ultrapure water at 4°C. The centrifugation conditions were 10000 g / min, 15 minutes each time. Finally, the mixture was resuspended in 0.1M phosphate buffer at pH 7.4 to obtain the selenium nanoparticle-cholera toxin B subunit complex suspension.

[0037] The concentration of dopamine hydrochloride added was 0.2 mg / ml, and it was combined with selenium nanoparticles in the following manner:

[0038] After the selenium nanoparticles were synthesized in a stock suspension, dopamine hydrochloride and selenium nanoparticles were incubated together in an alkaline buffer solution at pH 8.5 at 30°C for 4 hours, so that dopamine could self-polymerize on the surface of the selenium nanoparticles to form a polydopamine coating layer.

[0039] The loading method for curcumin was as follows: curcumin was dissolved in anhydrous ethanol to prepare a curcumin ethanol solution with a concentration of 3 mg / ml. Under light-protected conditions, the curcumin ethanol solution was added dropwise to the obtained selenium nanoparticle complex suspension, and the final concentration of ethanol was controlled not to exceed 5% of the total volume of the solution. Then, under room temperature and light-protected conditions, the mixture was magnetically stirred at 200 rpm for 12 hours. After loading was completed, the mixture was centrifuged at 4℃ and 13500 g / min for 25 minutes to remove unloaded free curcumin.

[0040] After centrifugation to remove unloaded free curcumin, the process includes a precipitation resuspension step: at 4°C, phosphate buffer is added to the selenium nanoparticle precipitate after free curcumin removal for resuspension, and the volume is adjusted to a final concentration of 2.5 mg Se / mL. The mixture is then placed on a magnetic stirrer and stirred continuously at 200 rpm for 15 minutes to ensure that the precipitate is fully dispersed in the buffer solution. The mixture is then transferred to a sterile centrifuge tube and centrifuged again at 9000 g / min for 7 minutes. The supernatant is discarded to remove any remaining undispersed particles. The resulting selenium nanoparticle resuspension is a uniformly dispersed intramuscular selenium nanoparticle formulation.

[0041] The phosphate buffer solution is a 0.05M, pH 7.4 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a total sodium ion concentration of 0.1M.

[0042] The surfactant was selected from Pluronic F-127 and was added as follows: at room temperature and in the dark, the surfactant was slowly added dropwise to the selenium nanoparticle suspension at a rate of 1.5 mL / min, and the mixture was continuously stirred at a speed of 400 rpm during the addition process. After the addition was completed, the mixture was stirred for another 20 minutes to ensure that the surfactant was evenly dispersed.

[0043] Before use, the selenium nanoparticle formulation is adjusted to pH 7.4 with sterile physiological saline and sterilized by passing it through a 0.22μm sterile filter membrane.

[0044] The selenium nanoparticle formulation is packaged in a pre-filled syringe in unit dose form, with a unit dose volume of 1.2 mL, and is administered via intramuscular injection.

[0045] Example 3: An intramuscular selenium nanoparticle formulation for the treatment of spinal cord injury, the selenium nanoparticle formulation being made from the following raw materials in parts by weight: 80 parts selenium nanoparticles, 15 parts cholera toxin B subunit, 10 parts dopamine hydrochloride, 8 parts curcumin, and 5 parts surfactant.

[0046] Selenium nanoparticles were prepared by the following method: 100 mg of selenocysteine ​​was added to 50 ml of water. Under magnetic stirring, the pH of the mixture was adjusted to 9.5 with 1.0 M NaOH solution. The mixture was then continuously shaken and reacted at 50 °C in the dark for 28 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed in deionized water at 10 °C for 24 hours. The dialysis solution was replaced every 8 hours. After dialysis, the liquid in the dialysis bag was centrifuged at 4 °C and 15000 g / min for 30 minutes. The supernatant was discarded, and the resulting precipitate was the selenium nanoparticles. The precipitate was resuspended in 0.5 M phosphate buffer solution with a pH of 7.6 to adjust the concentration to 10 mg Se / ml and stored at 4 °C for later use. The resulting resuspended solution was the selenium nanoparticle stock suspension.

[0047] The cholera toxin B subunit and selenium nanoparticles were covalently linked. The specific linking method was as follows: a stock suspension of selenium nanoparticles was washed three times with 0.1M 2-(N-morpholine) ethanesulfonic acid buffer. Then, it was mixed with 15 parts by weight of cholera toxin B subunit, 8 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4 parts by weight of N-hydroxysuccinimide in ultrapure water. The total volume of the reaction system was controlled at 15 ml. The reaction was carried out with gentle shaking at room temperature and in the dark for 3 hours. After the reaction was completed, the mixture was washed four times with ultrapure water at 4°C. The centrifugation conditions were 12000 g / min, 20 minutes each time. Finally, the mixture was resuspended in 0.2M phosphate buffer at pH 7.6 to obtain the selenium nanoparticle-cholera toxin B subunit complex suspension.

[0048] The concentration of dopamine hydrochloride added was 0.3 mg / ml, and it was combined with selenium nanoparticles in any of the following ways:

[0049] Dopamine hydrochloride was added to the selenium nanoparticle stock suspension, and the pH of the selenium nanoparticle stock suspension was adjusted to 9.5. The suspension was then incubated together at 37°C for 6 hours to allow dopamine to self-polymerize on the surface of the selenium nanoparticles to form a polydopamine coating layer.

[0050] The loading method for curcumin was as follows: curcumin was dissolved in anhydrous ethanol to prepare a curcumin ethanol solution with a concentration of 5 mg / ml. Under light-protected conditions, the curcumin ethanol solution was added dropwise to the obtained selenium nanoparticle complex suspension, and the final concentration of ethanol was controlled not to exceed 5% of the total volume of the solution. Then, under room temperature and light-protected conditions, the mixture was magnetically stirred at 300 rpm for 16 hours. After loading was completed, the mixture was centrifuged at 4℃ and 15000 g / min for 30 minutes to remove unloaded free curcumin.

[0051] After centrifugation to remove unloaded free curcumin, the process includes a precipitation resuspension step: at 4°C, phosphate buffer is added to the selenium nanoparticle precipitate after free curcumin removal for resuspension, and the volume is adjusted to a final concentration of 5.0 mg Se / mL. The mixture is then placed on a magnetic stirrer and stirred continuously at 300 rpm for 20 minutes to ensure that the precipitate is fully dispersed in the buffer solution. The mixture is then transferred to a sterile centrifuge tube and centrifuged again at 10000 g / min for 10 minutes. The supernatant is discarded to remove any remaining undispersed particles. The resulting selenium nanoparticle resuspension is a uniformly dispersed intramuscular selenium nanoparticle formulation.

[0052] The phosphate buffer solution is a 0.1M, pH 7.6 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a total sodium ion concentration of 0.2M.

[0053] The surfactant was selected from polyvinylpyrrolidone K30. The method of addition was as follows: under room temperature and light-protected conditions, the surfactant was slowly added dropwise to the selenium nanoparticle suspension at a rate of 2.0 mL / min, and the mixture was continuously stirred at a speed of 600 rpm during the addition process. After the addition was completed, the mixture was stirred for 30 minutes to ensure that the surfactant was evenly dispersed.

[0054] Before use, the selenium nanoparticle formulation was adjusted to pH 7.6 with sterile phosphate buffer and sterilized by passing it through a 0.22μm sterile filter membrane.

[0055] The selenium nanoparticle formulation is packaged in a vial in unit dose form, with a unit dose volume of 2.0 mL, and is administered via intramuscular injection.

[0056] Comparative Example 1: The difference between this comparative example and Example 1 is that no cholera toxin B subunit was added when preparing selenium nanoparticles in this comparative example.

[0057] Comparative Example 2 differs from Example 1 in that: in this comparative example, dopamine hydrochloride was not added for coating when preparing selenium nanoparticles.

[0058] Comparative Example 3 differs from Example 1 in that curcumin was not loaded during the preparation of the selenium nanoparticle suspension in this comparative example.

[0059] Comparative Example 4 differs from Example 1 in that the nanoparticle formulation prepared in this comparative example was not subjected to pH adjustment and sterilization treatment with phosphate buffer.

[0060] The performance of the selenium nanoparticle formulations prepared in Examples 1-3 and Comparative Examples 1-4 was tested. The test items and methods are as follows:

[0061] Particle size and distribution test: Take 3 mL of nanoparticle formulation, dilute it 100 times with deionized water, and use a dynamic light scattering particle size analyzer to determine its hydrated particle size. The test conditions are: temperature 25℃, equilibration time 120 seconds, and measurement angle 173°.

[0062] Zeta potential test: 2 mL of nanoparticle formulation was injected into the sample cell of the laser particle size analyzer and its surface Zeta potential was measured under constant temperature of 25℃. Each sample was measured three times and the average value was taken.

[0063] Encapsulation efficiency was tested by separating free curcumin from nanoparticles using high-speed centrifugation at 12,000 rpm for 30 minutes. The precipitate was collected and reconstituted with a small amount of phosphate buffer. The characteristic absorbance of curcumin was measured at 425 nm using a UV-Vis spectrophotometer, and the mass of the free drug was calculated based on a standard curve. and the quality of drugs encapsulated within particles Total amount of medicine used The encapsulation ratio is calculated using the following formula: .

[0064] For the in vitro release test, 1 mL of the nanoparticle formulation was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, clamped at both ends, and then placed in a beaker containing 20 mL of phosphate buffer solution at pH 7.4. The beaker was placed in a constant-temperature shaker and shaken at 37°C and 50 rpm. 2 mL of the sample was taken after 24 hours, and an equal volume of fresh release medium was added simultaneously. The absorbance was measured using a UV-Vis spectrophotometer, and the cumulative release rate was calculated.

[0065] The test data of the selenium nanoparticle formulations prepared in Examples 1-3 and Comparative Examples 1-4 are recorded in the table below:

[0066] Example 1 85.4±2.1 -21.5±1.2 92.5±1.8 68.4±3.2 Example 2 88.2±2.5 -20.8±1.5 91.2±2.1 70.1±2.9 Example 3 86.7±3.0 -21.0±1.3 90.5±2.3 69.7±3.5 Comparative Example 1 156.3±5.2 -8.4±0.9 45.2±4.5 95.6±4.1 Comparative Example 2 92.5±3.1 -10.2±1.1 75.4±3.2 82.3±3.7 Comparative Example 3 178.9±6.5 -5.6±0.8 38.7±5.1 98.2±2.8 Comparative Example 4 105.4±4.2 -12.8±1.4 68.9±3.6 89.5±3.3

[0067] Comparison and analysis of the data in the table show that the intramuscularly administered selenium nanoparticle formulations prepared using the processes in Examples 1-3 exhibit superior performance in key quality attributes compared to the formulations prepared using the processes in Comparative Examples 1-4. This indicates that using selenium nanoparticles as the delivery core, with their surface coated by a polydopamine layer, improves particle stability and biocompatibility, and reduces non-specific clearance in systemic circulation. Simultaneously, this polydopamine coating layer provides an active interface for functional modification; the covalently linked cholera toxin B subunit can recognize and bind to specific receptors in peripheral nerve endings, delivering curcumin-loaded selenium nanoparticles from the intramuscular injection site to the damaged spinal cord region. This solves the problems of poor targeting and insufficient accumulation at the lesion site in conventional nanoparticle formulations, enhancing the accuracy of treatment. The functional component selenium nanoparticles, combined with the loaded drug curcumin, construct a synergistic therapeutic system. Selenium nanoparticles possess antioxidant and anti-inflammatory activities, capable of neutralizing excessive harmful reactive oxygen species at the site of injury. Loaded curcumin exerts anti-inflammatory and neuroprotective effects; both are co-delivered via nanocarriers, producing a synergistic effect at the injury site. This achieves a synergistic intervention on oxidative stress and inflammatory responses following spinal cord injury, enhancing overall therapeutic efficacy. Added surfactants prevent nanoparticle aggregation during storage, maintaining their uniform nanoscale dispersion. The final formulation undergoes pH adjustment and aseptic filtration, forming a physicochemically stable, sterile suspension suitable for direct intramuscular injection. This ensures stability throughout the entire process from preparation and storage to administration, laying the foundation for clinical translation feasibility.

[0068] By comparing and analyzing the relevant data in the table, it can be seen that the intramuscular selenium nanoparticle formulation prepared by the process of the present invention has comprehensive advantages such as uniform particle size, good stability, high drug loading efficiency, and controllable release behavior.

[0069] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury, characterized in that: The selenium nanoparticle formulation is made from the following raw materials in parts by weight: 60-80 parts selenium nanoparticles, 5-15 parts cholera toxin B subunit, 5-10 parts dopamine hydrochloride, 2-8 parts curcumin, and 1-5 parts surfactant.

2. The intramuscularly administered selenium nanoparticle formulation for spinal cord injury treatment according to claim 1, characterized in that: The selenium nanoparticles were prepared by the following method: 50-100 mg of selenocysteine ​​was added to 20-50 ml of water. Under magnetic stirring, the pH of the mixture was adjusted to 8.5-9.5 with 0.1-1.0 M NaOH solution. The mixture was then continuously shaken and reacted at 50°C in the dark for 20-28 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3000-5000 Da and dialyzed in deionized water at 4-10°C for 12-24 hours, with the dialysis fluid replaced every 4-8 hours. After dialysis, the liquid in the dialysis bag was centrifuged at 4°C and 10000-15000 g / min for 15-30 minutes. The supernatant was discarded, and the resulting precipitate was the selenium nanoparticles. These were resuspended in 0.01-0.5 M phosphate buffer (pH 7.2-7.6) to adjust the concentration to 5-10 mg. Se / ml, stored at 4℃ for later use, the resulting resuspended solution is the selenium nanoparticle stock suspension.

3. The intramuscularly administered selenium nanoparticle formulation for spinal cord injury treatment according to claim 1, characterized in that: The cholera toxin B subunit is covalently linked to selenium nanoparticles. The specific linkage method is as follows: Take a stock suspension of selenium nanoparticles and wash it 1-3 times with 0.01-0.1M 2-(N-morpholine)ethanesulfonic acid buffer. Then, mix it with 5-15 parts by weight of cholera toxin B subunit, 4-8 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-4 parts by weight of N-hydroxysuccinimide in ultrapure water. Control the total volume of the reaction system to be 5-15 ml. Gently shake the mixture at room temperature and in the dark for 1-3 hours. After the reaction is completed, wash it 2-4 times with ultrapure water at 4°C. The centrifugation conditions are 8000-12000 g / min, 10-20 minutes each time. Finally, resuspend it with phosphate buffer with pH 7.2-7.6 and a concentration of 0.01-0.2M to obtain a selenium nanoparticle-cholera toxin B subunit complex suspension. This suspension is the selenium nanoparticle complex suspension.

4. The intramuscularly administered selenium nanoparticle formulation for spinal cord injury treatment according to claim 2, characterized in that: The concentration of dopamine hydrochloride added is 0.1-0.3 mg / ml, and it is combined with selenium nanoparticles in any of the following ways: Dopamine hydrochloride was added to the selenium nanoparticle stock suspension, and the pH of the selenium nanoparticle stock suspension was adjusted to 8.5-9.

5. The suspension was then incubated together at 25-37℃ for 2-6 hours to allow dopamine to self-polymerize on the surface of the selenium nanoparticles to form a polydopamine coating layer. After the selenium nanoparticles were synthesized in a stock suspension, dopamine hydrochloride and the selenium nanoparticles were incubated together in an alkaline buffer solution with a pH of 8.0-9.0 at 25-37°C for 2-6 hours, so that dopamine self-polymerized on the surface of the selenium nanoparticles to form a polydopamine coating layer.

5. The intramuscularly administered selenium nanoparticle formulation for spinal cord injury treatment according to claim 1, characterized in that: The loading method for curcumin is as follows: curcumin is dissolved in anhydrous ethanol to prepare a curcumin ethanol solution with a concentration of 1-5 mg / ml. Under light-protected conditions, the curcumin ethanol solution is added dropwise to the obtained selenium nanoparticle complex suspension, and the final concentration of ethanol is controlled not to exceed 5% of the total volume of the solution. Then, under room temperature and light-protected conditions, the mixture is magnetically stirred at a speed of 100-300 rpm for 8-16 hours. After loading is completed, the mixture is centrifuged at 4℃ and 12000-15000 g / min for 20-30 minutes to remove unloaded free curcumin.

6. A selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury according to claim 5, characterized in that: After centrifugation to remove unloaded free curcumin, the process further includes a precipitate resuspension step: at 4°C, phosphate buffer is added to the selenium nanoparticle precipitate after removing free curcumin for resuspension, and the volume is adjusted to a final concentration of 1.0-5.0 mg Se / mL. Then, the precipitate is placed on a magnetic stirrer and stirred continuously at 100-300 rpm for 10-20 minutes to ensure that the precipitate is fully dispersed in the buffer solution. The mixture was then transferred to a sterile centrifuge tube and centrifuged again at 8000-10000 g / min for 5-10 minutes. The supernatant was discarded to remove any remaining undispersed particles. The resulting selenium nanoparticle resuspension was a uniformly dispersed intramuscular selenium nanoparticle formulation.

7. A selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury according to claim 6, characterized in that: The phosphate buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration of 0.01-0.1M and a pH of 7.2-7.6, and its total sodium ion concentration is 0.02-0.2M.

8. A selenium nanoparticle formulation for the treatment of spinal cord injury via intramuscular administration according to claim 6, characterized in that: The surfactant is selected from one of polysorbate-80, Pluronic F-127, polyvinylpyrrolidone K30, and sodium dodecyl sulfate. The method of addition is as follows: under room temperature and light-protected conditions, the surfactant is slowly added dropwise to the selenium nanoparticle suspension at a rate of 0.5-2.0 mL / min, and the mixture is continuously stirred at a speed of 200-600 rpm during the addition process. After the addition is completed, stirring is continued for 10-30 minutes to ensure that the surfactant is evenly dispersed.

9. A selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury according to claim 1, characterized in that: Before use, the selenium nanoparticle formulation is adjusted to a pH of 7.2-7.6 with sterile phosphate buffer or physiological saline and sterilized by passing it through a 0.22μm sterile filter membrane.

10. A selenium nanoparticle formulation for intramuscular administration in the treatment of spinal cord injury according to claim 1, characterized in that: The selenium nanoparticle formulation is packaged in a vial or pre-filled syringe in unit dose form, with a unit dose volume of 0.5-2.0 mL, and is administered via intramuscular injection.