L-menthol-based double-acting phase-change carrier-free nano-particle as well as preparation method and application thereof
By preparing L-menthol-based dual-action phase change carrier-free nanoparticles, the problems of rapid metabolism of L-menthol and easy clearance of carrier-free nanomedicines were solved, enabling precise treatment and repair of spinal cord injury.
Patent Information
- Application Number
- CN202511323442.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-16
- Publication Date
- 2026-01-02
AI Technical Summary
In existing technologies, the small molecule properties and rapid metabolism of L-menthol limit its therapeutic precision in the treatment of spinal cord injury, and carrier-free nanomedicine platforms are easily cleared and released uncontrollably, affecting the therapeutic effect.
A carrier-free phase change nanoparticle preparation method based on L-menthol was adopted. By combining hydrogenated lecithin, lauric acid and L-menthol, and combining anti-inflammatory drugs such as methylprednisolone, nanoparticles with excellent biocompatibility and temperature dual response were formed, enabling in-situ injection controlled release.
This approach achieves reduced nanoparticle clearance rate, controlled release, regulation of the immune microenvironment in spinal cord injury, and repair of the blood-spinal cord barrier, thus effectively treating spinal cord injury.
Smart Images
Figure CN121243082A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biomedical materials, and particularly relates to a dual-acting phase change carrier-free nanoparticle based on L-menthol, a preparation method thereof and application thereof. BACKGROUND
[0002] The main obstacles hindering functional recovery after injury include insufficient and delayed reestablishment of the blood-spinal cord barrier (BSCB) and excessive inflammatory responses, and in order to solve these problems, the method of mild cold stimulation therapy in coordination with immunomodulation is very promising for the repair of tissues after spinal cord injury.
[0003] The glucocorticoid drug methylprednisolone (MP) has been used in the clinical treatment of spinal cord injury due to its strong anti-inflammatory and immunosuppressive effects. Since low-temperature therapy can play a therapeutic role by activating the TRPM8 channel, but there are disadvantages such as instability of the thermal gradient affecting the accuracy of targeting and tissue damage caused by temperature fluctuations, the use of the TRPM8 channel activator L-menthol is considered to be an alternative strategy, but the small molecule characteristics and rapid metabolism of L-menthol limit its treatment accuracy, and an advanced delivery platform is needed.
[0004] In recent years, due to the emerging carrier-free nanomedicine technology which can solve the safety problems such as unpredictable central nervous system biodistribution and metabolic complications of traditional intrathecal nanocarriers, carrier-free nanomedicine is increasingly used in various fields including spinal cord injury, however, the existing platform usually forms solid nanoparticles which are easily quickly cleared by macrophages and released uncontrollably.
[0005] In summary, improving the characteristics of the L-menthol and methylprednisolone co-delivery platform to reduce its clearance rate and controlled release, etc. has important significance and prospects for the treatment of spinal cord injury. SUMMARY
[0006] The present application provides a preparation method of a dual-acting phase change carrier-free nanoparticle based on L-menthol and application thereof, which has excellent biocompatibility, ROS and temperature dual responsiveness and other characteristics.
[0007] To solve the above technical problems, the technical scheme of the present application is as follows:
[0008] A preparation method of a dual-acting phase change carrier-free nanoparticle based on L-menthol, comprising the following steps:
[0009] Step 1: Disperse hydrogenated lecithin in an alcohol-water mixed solvent and heat to obtain a hydrogenated lecithin suspension;
[0010] Step 2: Dissolve lauric acid and L-menthol in organic solvent completely;
[0011] Step 3: Dissolve anti-inflammatory drug in organic solvent until the solution is clear and transparent, obtaining a drug solution;
[0012] Step 4: Mix the anti-inflammatory drug solution of Step 3 with the solution of Step 2 uniformly, and then drop into the hydrogenated lecithin suspension obtained in Step (1), and stir vigorously for 2-5 minutes;
[0013] Step 5: After completion of stirring, quickly put into ice water and cool for 2-5 minutes, then take out, and after the turbid solution returns to room temperature, stir for 1-2 minutes;
[0014] Step 6: Filter the turbid solution through a 0.22 μm microporous filter membrane, and stop rotary evaporation when the remaining liquid volume is 10-20% of the original volume, obtaining the nanoparticles.
[0015] Preferably, the mass-volume ratio of the hydrogenated lecithin to the alcohol-water mixed solvent in Step 1 is 1 mg: 0.5-2 mL;
[0016] The alcohol in the alcohol-water mixed solvent is one or more of methanol, ethanol, and isopropanol, and the volume percentage of alcohol in the alcohol-water mixed solvent is 2-6%;
[0017] The temperature of the heating is 45-55°C.
[0018] Preferably, in Step 2, the mass ratio of lauric acid to L-menthol is 1:0.7-0.8;
[0019] The organic solvent is one or more of methanol, ethanol, isopropanol, and ethyl acetate;
[0020] The amount of the organic solvent is such that the total concentration of lauric acid and L-menthol is 3-4 mg / ml, and the mass ratio of lauric acid to L-menthol has a relatively important influence on the properties and therapeutic effect of the nanoparticles, and too large or too small mass ratio will affect the properties of the nanoparticles.
[0021] Preferably, in Step 3, the anti-inflammatory drug is one or more of methylprednisolone, dexamethasone, or betamethasone, and further preferably methylprednisolone;
[0022] The organic solvent is one or more of dimethyl sulfoxide, methanol, ethanol, and ethyl acetate, and further preferably dimethyl sulfoxide, and the concentration of the methylprednisolone solution is 80-100 mg / ml, and the concentration of methylprednisolone also has a relatively large influence on the therapeutic effect of the nanoparticles.
[0023] As preferred, in step 4, the solution of step 3 is mixed with the solution of step 2 first, and the rotating speed of the stirring is 1000-2000 rpm.
[0024] As preferred, in step 6, the temperature of the rotary evaporation is 50-60℃, and the residual liquid volume after the rotary evaporation is 10-20% of the original volume.
[0025] As preferred, in step 6, the solution needs to be filtered through a 0.22 μm microporous filter before the rotary evaporation.
[0026] The application also provides an L-menthol-based dual-acting phase change carrier-free nanoparticle, which is obtained according to the above preparation method.
[0027] The application also provides the use of the nanoparticle in the preparation of a medicine, characterized in that the medicine is used for treating spinal cord injury.
[0028] As preferred, when the medicine is used for treating spinal cord injury, the nanoparticle is injected in situ.
[0029] Compared with the prior art, the application has the following beneficial effects:
[0030] The nanoparticle obtained by the application has excellent biocompatibility, ROS and temperature dual responsiveness, etc., and can be injected in situ to reduce its clearance rate and control the release, adjust the immune microenvironment of spinal cord injury and repair the blood-spinal cord barrier, so as to achieve the effect of treating spinal cord injury. BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 Figure 1 is a transmission electron microscope morphology diagram of the NP@MP nanoparticle in Example 1;
[0032] Figure 2 Figure 3 is a particle size distribution diagram of the NP nanoparticle formed by combining lauric acid and L-menthol in different proportions in Example 1;
[0033] Figure 3 Figure 5 is a particle size change diagram of the NP@MP nanoparticle loaded with different concentrations of methylprednisolone for two days in Example 1;
[0034] Figure 4 Figure 7 is an FT-IR characterization diagram of lauric acid, L-menthol, methylprednisolone (MP) and the nanoparticle in Example 1.
[0035] Figure 5 Figure 9 is a differential scanning calorimetry analysis curve of the nanoparticle in Example 1.
[0036] Figure 6 Figure 11 is the in vitro release rate of the nanoparticle under the stimulation of hydrogen peroxide in Example 2.
[0037] Figure 7 Fluorescent images of live and dead cell staining of PC-12 cells treated with different concentrations of nanoparticles in Example 3.
[0038] Figure 8 Detection of blood-spinal cord barrier integrity in Example 4. (a) Actual image of spinal cord tissue of mice after injection of Evans blue. (b) Statistical chart of Evans blue concentration in spinal cord tissue. (c) Fluorescent image of Evans blue in injured spinal cord.
[0039] Figure 9 Inflammation in the injury area of the sham operation group, nanoparticle group and blank group in Example 5.
[0040] Figure 10 Tissue recovery of the sham operation group, nanoparticle group and blank group in Example 6. DETAILED DESCRIPTION
[0041] The present application is described in detail below with reference to the accompanying drawings and examples.
[0042] Example 1
[0043] The preparation method of the L-menthol-based dual-action phase change carrier-free nanoparticles in the present embodiment is as follows:
[0044] Step 1: Weigh 15 mg of hydrogenated lecithin and disperse it in 15 ml of 4% methanol solution, and heat it to warm it to 50°C.
[0045] Step 2: Weigh about 6.7 mg of lauric acid and 5.3 mg of L-menthol, and add 3 ml of methanol to completely dissolve them.
[0046] Step 3: Weigh 9 mg of methylprednisolone and dissolve it in 90 μl of dimethyl sulfoxide until the solution is clear and transparent.
[0047] Step 4: First, mix the solution of Step 3 with the solution of Step 2 evenly, then slowly drop it into the 50°C hydrogenated lecithin suspension, and stir vigorously for 2 minutes.
[0048] Step 5: After stirring is completed, quickly place it in ice water to cool for 2 minutes, then take it out, and after the turbid solution returns to room temperature, stir it for 2 minutes.
[0049] Step 6: Pass the turbid solution through a 0.22 μm microporous filter membrane, and rotary evaporate it at 50°C until the remaining liquid is 3 mL to stop, obtaining the NP@MP3 nanoparticles. According to the same steps, NP nanoparticles can be obtained without adding methylprednisolone.
[0050] The obtained NP and NP@MP nanoparticles are detected, Figure 1 is the transmission electron microscope morphology of the NP@MP nanoparticles,Figure 2 Figure 6 is a graph showing the particle size distribution of NP nanoparticles formed by lauric acid and L-menthol in different proportions, Figure 3 Figure 7 is a graph showing the particle size change of NP@MP nanoparticles loaded with different concentrations of methylprednisolone over two days, wherein NP@MP1, NP@MP2, NP@MP3, NP@MP4, and NP@MP5 represent methylprednisolone concentrations of 25 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, and 175 mg / ml, respectively, Figure 4 Figure 8 is an FT-IR characterization graph, Figure 5 Figure 9 is a differential scanning calorimetry analysis curve. Figure 2 The results show that the particle size of lauric acid and L-menthol is about 100 nm and is uniformly distributed at a ratio of 1:0.8. Figure 3 The results show that when the concentration of methylprednisolone is 100 mg / ml, the prepared nanoparticles have a large drug loading capacity and strong stability. At the same time, Figure 4 The FT-IR characterization graphs of lauric acid, L-menthol, and methylprednisolone are also listed.
[0051] Example 2
[0052] NP@MP3 nanoparticle in vitro release experiment: 4-(1,2,2-triphenylvinyl) benzoic acid was used as a methylprednisolone substitute to evaluate the in vitro release kinetics. Methylprednisolone was replaced with 4-(1,2,2-triphenylvinyl) benzoic acid, and nanoparticles were prepared according to the same method and divided into two equal parts. One was placed in pure water, and the other was placed in an equal amount of 50 mM hydrogen peroxide. The external liquid was drawn at certain time intervals and supplemented with an equal amount of external liquid. The external liquid drawn at different time intervals was measured at a wavelength of 360 nm. The standard curve was compared, and the cumulative release in vitro was calculated. The results are shown in Figure 6 The results show that the NP@MP nanoparticles release faster and have a larger release amount in the presence of hydrogen peroxide, indicating a certain responsiveness to hydrogen peroxide.
[0053] Example 3
[0054] NP@MP3 nanoparticle live and dead cell staining of PC-12 cells: After the PC-12 cells were plated and ready for experiments, a certain amount of NP@MP nanoparticles was added to the liquid volume of the culture medium to prepare a nanoparticle dispersion solution with a certain concentration. After the PC-12 cells were co-cultured with different concentrations of nanoparticles for 24 h, live and dead cell fluorescent dyes were added. The results are shown in Figure 7 The results show that the different concentrations of nanoparticles prepared have no obvious toxic side effects on PC-12 cells, indicating good biocompatibility.
[0055] Example 4
[0056] Evans blue leakage experiment: After the mice were anesthetized with 1% pentobarbital and the back fur was removed, the back skin was cut and the vertebral plate of the T10 segment was found. After prying the vertebral plate with a tool, the exposed spinal cord could be seen. A precision impactor was used to create a bruise model. The following sham operation group followed the same steps but was not bruised. According to whether the group was injected with 7 μL of NP@MP nanoparticles in situ, after 3 days of feeding, the blank group and the NP@MP3 group were injected with 1% Evans blue solution through the tail vein. After 3 hours, the mice were anesthetized and perfused through the heart. The spinal cord tissue was removed. One spinal cord was randomly selected from each group, placed in PBS, and homogenized with a homogenizer. After 24 hours at room temperature, the absorbance was measured. The remaining spinal cord was embedded and then the fluorescence image was taken. The results are shown in Figure 8 The results show that the Evans blue leakage in the NP@MP3 nanoparticle treatment group is significantly reduced, indicating that the nanoparticle is beneficial to the repair of the blood-spinal cord barrier.
[0057] Example 5
[0058] According to the modeling and dosing in Example 4, after feeding the mice for 7 days, the spinal cord tissue of the sham operation group, the blank group and the NP@MP3 group was taken out for the same treatment. After embedding and ice cutting, the PBS was used to wash away the impurities around the tissue, and 5% BSA was used for blocking. The diluted CD206 / CD86 primary antibody was added, and incubated overnight at 4°C. After washing with PBS, the corresponding species secondary antibody was added, and incubated at 37°C for 1 hour. After washing with PBS, fluorescence imaging was performed. The results are shown in Figure 9 The results show that the CD206 intensity in the NP@MP3 nanoparticle treatment group is significantly higher than that in the blank group, and the CD86 is lower than that in the blank group, indicating that the nanoparticle has anti-inflammatory effect
[0059] Example 6
[0060] According to the modeling and dosing in Example 4, after feeding the mice for 28 days, the spinal cord tissue of the sham operation group, the blank group and the NP@MP3 group was taken out for the same treatment. After embedding and ice cutting, the tissue was stained according to the operation of the hematoxylin / eosin staining kit instruction manual. The recovery of the damaged tissue was observed. The results are shown in Figure 10 The results show that after treatment with NP@MP3 nanoparticles, the tissue recovery is significantly better than that of the blank group, and the tissue structure is more complete, approaching the sham operation group, indicating that the nanoparticle has a significant effect on spinal cord injury treatment.
Claims
1. A method for preparing carrier-free phase change nanoparticles based on L-menthol dual-action phase change, characterized in that, Includes the following steps: Step 1: Disperse hydrogenated lecithin in an alcohol-water mixed solvent and heat to obtain a hydrogenated lecithin suspension; Step 2: Add lauric acid and L-menthol to an organic solvent to dissolve them completely; Step 3: Dissolve the anti-inflammatory drug in an organic solvent until the solution is clear and transparent to obtain a drug solution; Step 4: First, mix the anti-inflammatory drug solution such as methylprednisolone, dexamethasone or betamethasone from step 3 with the solution from step 2, and then drop it into the hydrogenated lecithin suspension obtained in step (1), and stir vigorously for 2 to 5 minutes; Step 5: After stirring, quickly place the solution in ice water to cool for 2-5 minutes, then remove it and stir for 1-2 minutes after the turbid solution has returned to room temperature. Step 6: Pass the turbid solution through a 0.22μm microporous membrane and rotary evaporate until the remaining liquid volume is 10-20% of the original volume to obtain the nanoparticles.
2. The method for preparing carrier-free L-menthol-based dual-action phase change nanoparticles according to claim 1, characterized in that: The mass-to-volume ratio of hydrogenated lecithin to the alcohol-water mixed solvent in step 1 is 1 mg: 0.5–2 mL; The alcohol in the alcohol-water mixed solvent is one or more of methanol, ethanol, and isopropanol, and the volume percentage of alcohol in the alcohol-water mixed solvent is 2-6%. The heating temperature is 45-55℃.
3. The method for preparing carrier-free L-menthol-based dual-action phase change nanoparticles according to claim 1, characterized in that: In step 2, the mass ratio of lauric acid to L-menthol is 1:0.7 to 0.8; The organic solvent is one or more of methanol, ethanol, isopropanol, and ethyl acetate; The amount of organic solvent used results in a total concentration of lauric acid and L-menthol of 3–4 mg / ml.
4. The method for preparing L-menthol-based dual-action phase change carrier-free nanoparticles according to claim 1, characterized in that: In step 3, the anti-inflammatory drug is one or more of methylprednisolone, dexamethasone, or betamethasone; The organic solvent is one or more of dimethyl sulfoxide, methanol, ethanol, and ethyl acetate, and the concentration of the anti-inflammatory drug solution is 80-100 mg / ml.
5. The method for preparing L-menthol-based dual-action phase change carrier-free nanoparticles according to claim 1, characterized in that: In step 4, the solution from step 3 is first mixed with the solution from step 2 until they are homogeneous, and the stirring speed is 1000-2000 rpm.
6. The method for preparing L-menthol-based dual-action phase change carrier-free nanoparticles according to claim 5, characterized in that: In step 6, the rotary evaporation temperature is 50-60℃, and the volume of the residual liquid after rotary evaporation is 10-20% of the original volume.
7. The method for preparing L-menthol-based dual-action phase change carrier-free nanoparticles according to claim 6, characterized in that: In step 6, the material needs to be filtered through a 0.22μm microporous membrane before rotary evaporation.
8. A carrier-free phase change nanoparticle based on L-menthol, obtained by the preparation method according to any one of claims 1 to 7.
9. The application of L-menthol-based dual-action phase change carrier-free nanoparticles according to claim 8 in drug preparation, characterized in that, The drug is used to treat spinal cord injury.
10. The application according to claim 9, characterized in that, When the drug is used to treat spinal cord injury, the nanoparticles are administered via in-situ injection.