A rapamycin-loaded prussian blue targeted nanoparticle, a preparation method thereof and application thereof in a spinal cord injury treatment drug

Abstract

The application provides a preparation method of rapamycin-loaded Prussian blue targeted nanoparticles, which comprises the following steps: (1) preparing Prussian blue nanoparticles; (2) preparing hollow mesoporous Prussian blue nanoparticles; (3) preparing rapamycin-loaded hollow mesoporous Prussian blue; (4) preparing rapamycin-loaded Prussian blue targeted nanoparticles: polyethylene glycol and activatable cell-penetrating peptides are dissolved in ultrapure water, a catalyst N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added and stirred, then dialysis is performed, the rapamycin-loaded hollow mesoporous Prussian blue prepared in step (3) is added into the dialysate, stirring is performed, centrifugation is performed, and rapamycin-loaded Prussian blue targeted nanoparticles are obtained. The rapamycin is loaded into the hollow mesoporous Prussian blue, and the rapamycin-loaded Prussian blue is targetedly modified, so that the drug delivery efficiency and the targeting property are improved, and the effect is better.

Description

Technical Field

[0001] This invention relates to the field of spinal cord injury treatment technology, specifically to a rapamycin-loaded Prussian blue targeted nanoparticle, a method for preparing the rapamycin-loaded Prussian blue targeted nanoparticle, and its application in spinal cord injury treatment drugs. Background Technology

[0002] Spinal cord injury (SCI) is a neurological disorder that has become a heavy burden on various countries and regions due to its high disability and mortality rates. Traumatic factors, such as traffic accidents and falls, are the main causes of SCI, accounting for more than half of the precipitating factors. Based on the pathological process, SCI can be classified into primary and secondary injuries. Secondary injuries, particularly those causing inflammation leading to persistent cell death, demyelination, and scar formation, are also major contributing factors, triggering a cascade of damage and inducing further secondary injuries. Secondary SCI is a complex process involving the generation of large amounts of reactive oxygen species due to ischemia and hypoxia, as well as a local inflammatory outbreak at the injury site. Neuroinflammation also plays a crucial role in secondary SCI. Direct damage to the blood-spinal cord barrier and the excessive expression of matrix metalloproteinases exacerbate the disruption of the blood-spinal cord barrier, leading to the recruitment of numerous peripheral immune cells, such as neutrophils, monocytes, macrophages, and T cells, to the central nervous system.

[0003] Spinal cord injury remains one of the most serious diseases of the central nervous system, urgently requiring the development of effective treatment strategies. From the pathological process of spinal cord injury, primary spinal cord injury is mainly caused by direct damage from mechanical external forces, such as direct neuronal death and disruption of blood vessels and the blood-spinal barrier. This can exacerbate cascade damage and induce secondary spinal cord injury, including ischemia and hypoxia leading to excessive reactive oxygen species and neuroinflammation at the site of injury in the spinal cord tissue. Direct damage to the blood-spinal barrier and increased expression of matrix metalloproteinases further exacerbate its disruption, resulting in the recruitment of a large number of peripheral immune cells to the central nervous system, such as neutrophils, monocytes, macrophages, and T cells. Consequently, innate immune cells in the central nervous system, such as microglia and astrocytes, are overactivated, further amplifying inflammation and inducing neuronal damage. Currently, drug therapy, such as cyclooxygenase inhibitors, minocycline, granulocyte colony-stimulating factor, chondroitin ABC, and neuroimmunoprotein ligands, is used for anti-inflammatory treatment of spinal cord injury. In addition, various nanomedicine delivery systems, including polymers, iron oxide, gold, and quantum dots, are also used to treat spinal cord injuries by altering immune responses.

[0004] With the development of nanotechnology, many advances in drug therapy will overcome the current shortcomings in spinal cord injury treatment. Various biomaterial-based therapeutic strategies have been developed to target excessive oxidative stress and neuroinflammation following spinal cord injury. Nanozymes with antioxidant properties have been explored for the treatment of central nervous system diseases such as spinal cord injury, stroke, traumatic brain injury, and Parkinson's disease. Nanozymes possess characteristics such as stable enzyme-like activity, high antioxidant activity, good physiological stability, and ease of synthesis. However, existing nanomedicine delivery systems suffer from low drug delivery efficiency, inability to penetrate the blood-spinal cord barrier, poor stability, and poor biocompatibility, making it difficult to achieve effective treatment of spinal cord injury. Therefore, Prussian blue nanoparticles with antioxidant properties, modified with cell-activating transmembrane peptides, and loaded with the anti-inflammatory drug rapamycin, are used to improve their in vivo targeting and exert therapeutic effects on spinal cord injury.

[0005] Based on this, the present invention provides a method for preparing Prussian blue targeted nanoparticles loaded with rapamycin. By structurally designing Prussian blue to form a mesoporous structure, rapamycin is loaded into hollow mesoporous Prussian blue and simultaneously targeted modification is performed, which improves the delivery efficiency and targeting of rapamycin and enhances the drug's efficacy. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Prussian blue-loaded rapamycin-loaded targeted nanoparticles, which solves the problem of low drug delivery efficiency in existing drug delivery systems.

[0007] The present invention also aims to provide rapamycin-loaded Prussian blue targeted nanoparticles prepared by the above method.

[0008] Another objective of this invention is to provide the application of the above-mentioned rapamycin-loaded Prussian blue targeted nanoparticles.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for preparing Prussian blue-loaded rapamycin-based targeted nanoparticles includes the following steps:

[0011] (1) Preparation of Prussian blue nanoparticles;

[0012] (2) Preparation of hollow mesoporous Prussian blue nanoparticles;

[0013] (3) Preparation of hollow mesoporous Prussian blue loaded with rapamycin: Dissolve rapamycin in anhydrous ethanol, add it to the anhydrous ethanol solution of hollow mesoporous Prussian blue nanoparticles, stir and centrifuge, retain the precipitate, and obtain hollow mesoporous Prussian blue loaded with rapamycin.

[0014] (4) Preparation of rapamycin-loaded Prussian blue targeted nanoparticles: Polyethylene glycol and cell-penetrating peptides were dissolved in ultrapure water, and N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and stirred. Then, dialysis was performed. Hollow mesoporous Prussian blue loaded with rapamycin prepared in step (3) was added to the dialysis solution, stirred, and centrifuged to obtain rapamycin-loaded Prussian blue targeted nanoparticles.

[0015] In this invention, the mass-to-volume ratio of rapamycin to anhydrous ethanol in step (3) is 0.1–1 mg / mL; the mass-to-volume ratio of hollow mesoporous Prussian blue nanoparticles to anhydrous ethanol is 1–10 mg / mL.

[0016] Furthermore, in step (3), the mass-to-volume ratio of rapamycin to anhydrous ethanol is 0.5 mg / mL; and the mass-to-volume ratio of hollow mesoporous Prussian blue nanoparticles to anhydrous ethanol is 5 mg / mL.

[0017] In this invention, the mass-to-volume ratio of polyethylene glycol to ultrapure water in step (4) is 0.1–1 mg / mL; the mass-to-volume ratio of cell-penetrating peptide to ultrapure water is 0.1–1 / mL.

[0018] Furthermore, in step (4), the mass-to-volume ratio of polyethylene glycol to ultrapure water is 0.5 mg / mL; the mass-to-volume ratio of the cell-penetrating peptide to ultrapure water is 0.25 mg / mL.

[0019] In this invention, the amounts of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in step (4) are calculated at 0.1 mol per milliliter (mL).

[0020] In this invention, the molecular weight cutoff for dialysis in step (4) is 5000 Da.

[0021] In step (4), the product is stored at 4°C.

[0022] The present invention can be improved in the following way: step (1) Prussian blue nanoparticles can be prepared by microwave synthesis or hydrothermal method.

[0023] In this invention, Prussian blue nanoparticles are prepared by microwave synthesis. The specific process is as follows: potassium hexacyanoferrate(II) trihydrate and polyvinylpyrrolidone are weighed and dissolved in ultrapure water. Dilute hydrochloric acid solution is added and stirred until a clear solution is obtained. The reaction is carried out in a microwave reactor, centrifuged and resuspended, and dried to obtain Prussian blue nanoparticles.

[0024] Furthermore, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 5-7:1 mg / mL; the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.1-0.3 g / mL.

[0025] Preferably, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 6.585:1 mg / mL; and the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.15 g / mL.

[0026] Furthermore, the concentration of dilute hydrochloric acid is 0.05–0.4 mol / L. More preferably, it is 0.2 mol / L.

[0027] Furthermore, in the microwave reactor, the reaction temperature is 70-90℃ and the reaction time is 10-40 min.

[0028] Preferably, in the microwave reactor, the reaction temperature is 80°C and the reaction time is 20 min.

[0029] In this invention, Prussian blue nanoparticles are prepared by a hydrothermal method. The specific process is as follows: potassium hexacyanoferrate(II) trihydrate and polyvinylpyrrolidone are weighed and dissolved in ultrapure water. Dilute hydrochloric acid solution is added, and the mixture is stirred until a clear solution is obtained. The mixture is heated to carry out the reaction, and then centrifuged to obtain Prussian blue nanoparticles.

[0030] Furthermore, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 5-7:1 mg / mL; the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.1-0.3 g / mL.

[0031] Preferably, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 6.585:1 mg / mL; and the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.15 g / mL.

[0032] Further, the concentration of dilute hydrochloric acid is 0.01–0.05 mol / L. More preferably, it is 0.02 mol / L.

[0033] Furthermore, the heating and reaction temperature is 70-90℃, and the reaction time is 20-28h.

[0034] Preferably, the heating temperature for the reaction is 80°C, and the reaction time is 24 hours.

[0035] In some embodiments of the present invention, the specific process of preparing hollow mesoporous Prussian blue nanoparticles in step (2) is as follows: polyvinylpyrrolidone and the Prussian blue nanoparticles prepared in step (1) are dissolved in hydrochloric acid, and then the reaction is carried out in a polytetrafluoroethylene reactor. After the reaction is completed, the temperature is lowered and centrifuged to obtain a dark blue precipitate, which is the hollow mesoporous Prussian blue nanoparticles.

[0036] Further, the mass-to-volume ratio of polyvinylpyrrolidone to hydrochloric acid is 2.5–7.5 mg / mL; the mass-to-volume ratio of Prussian blue nanoparticles to hydrochloric acid is 0.5–1.5 mg / mL.

[0037] Preferably, the mass-to-volume ratio of polyvinylpyrrolidone to hydrochloric acid is 5 mg / mL; and the mass-to-volume ratio of Prussian blue nanoparticles to hydrochloric acid is 1 mg / mL.

[0038] Furthermore, the hydrochloric acid concentration is 1.5–2.5 mol / L; preferably 2 mol / L.

[0039] Furthermore, in the polytetrafluoroethylene reactor, the reaction temperature is 120–160°C, and the reaction time is 3–5 hours.

[0040] Preferably, the reaction temperature is 140°C and the reaction time is 4 hours.

[0041] The present invention can also be improved as follows: the centrifugation conditions are a centrifuge speed of 10,000 to 12,000 rpm and centrifugation at 10°C to 20°C for more than 10 minutes.

[0042] A type of Prussian blue-targeted nanoparticle loaded with rapamycin was prepared by the above method.

[0043] Furthermore, the Prussian blue in the loaded rapamycin Prussian blue targeted nanoparticles is ferriferric ferrocyanide with the chemical formula Fe4[Fe(CN)6]3.

[0044] The above-mentioned application of rapamycin-loaded Prussian blue-targeted nanoparticles in the preparation of drugs for treating spinal cord injury.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention provides a method for preparing rapamycin-loaded Prussian blue targeted nanoparticles. By modifying the structure of Prussian blue nanoparticles to construct a hollow mesoporous structure, rapamycin is loaded into the mesoporous Prussian blue, and the Prussian blue is targeted modified to obtain rapamycin-loaded Prussian blue targeted nanoparticles, which improves drug delivery efficiency and targeting, and has a better effect.

[0047] (2) The present invention uses Prussian blue as a drug carrier for treating spinal cord injury by loading rapamycin Prussian blue targeted nanoparticles. This overcomes the disadvantages of clinical rapamycin, such as large systemic toxic side effects and low utilization rate. In in vitro experiments, it has shown good antioxidant and anti-inflammatory activities.

[0048] (3) The present invention synthesizes Prussian blue through a microwave reactor, which has a shorter reaction time compared to the hydrothermal method. The nanomaterials synthesized by the microwave reactor have a higher yield and more uniform size.

[0049] (4) This invention explores different reaction temperatures to find the optimal temperature for preparing Prussian blue nanoparticles. In addition, by targeting and modifying Prussian blue, the targeting of diseased tissues in mice is improved. Through cell experiments and spinal cord injury mouse experiments, we found that rapamycin-loaded Prussian blue-targeted nanoparticles can effectively reverse the damage caused by oxidative stress, thereby achieving the functions of protecting neurons and promoting motor recovery, and can be used as a drug for treating spinal cord injury. Attached Figure Description

[0050] Figure 1 TEM images of Prussian blue (PB) prepared by hydrothermal method (A) or microwave synthesis method (B);

[0051] Figure 2 This is a graph showing the yield of Prussian blue (PB) prepared by hydrothermal or microwave synthesis methods.

[0052] Figure 3 These are TEM images of the prepared HMPB and RHPA nanoparticles;

[0053] Figure 4 The graph shows the particle size and potential results of the prepared HMPB and RHPA nanoparticles.

[0054] Figure 5 These are the X-ray diffraction patterns of PB, HMPB, and RH;

[0055] Figure 6 The ABTS free radical scavenging ability of targeted modified hollow mesoporous Prussian blue nanoparticles loaded with rapamycin RHPA (A) and rapamycin Rapa (B) was detected.

[0056] Figure 7 This is a graph showing the ability of PB and RHPA to scavenge hydroxyl radicals (A) or superoxide anions (B) using electron spin resonance (EPR) spectroscopy.

[0057] Figure 8 The images show the cell survival of human neuroblastoma cells after co-incubation with different concentrations of PB, HMPB, RHPA (A) or Rapa (B), and the cell survival rate of human neuroblastoma cells after treatment with RHPA using a glucose-oxygen deprivation model (C).

[0058] Figure 9 This is a graph showing (a) and (b) mitochondrial membrane potential of human neuroblastoma cells after treatment with a glucose-oxygen deprivation model, detected by flow cytometry.

[0059] Figure 10 Behavioral BMS scores of mice after treatment with HMPB, Rapa, RH, and RHPA in a spinal cord injury model.

[0060] Figure 11 Behavioral rotarod time plots of mice treated with HMPB, Rapa, RH, and RHPA in a spinal cord injury model.

[0061] Figure 12 Nissl staining of motor neurons in the anterior horn of the spinal cord in a mouse model of spinal cord injury after RHPA treatment;

[0062] Figure 13 Immunofluorescence staining of M1 macrophages in the spinal cord injury area of ​​mice after RHPA treatment in a spinal cord injury model. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention.

[0064] Example 1

[0065] A method for preparing Prussian blue-loaded rapamycin-based targeted nanoparticles includes the following steps:

[0066] (1) Preparation of Prussian blue nanoparticles by hydrothermal and microwave synthesis methods

[0067] a. Hydrothermal method: Under normal temperature and pressure (15-35℃, 1 standard atmosphere), take 3g of polyvinylpyrrolidone and 131.7mg of potassium ferricyanide and dissolve them in 20mL of ultrapure water. Add 20mL of 0.02M hydrochloric acid and stir for 30 minutes. React at 80 degrees Celsius for 24 hours. After the reaction is cooled, centrifuge at 12000rpm and 10 degrees Celsius for 15 minutes in a low-temperature high-speed centrifuge to obtain a deep blue precipitate, Prussian blue (PB).

[0068] b. Microwave Synthesis Method: Under normal temperature and pressure (15–35℃, 1 standard atmosphere), dissolve 1.12 g of polyvinylpyrrolidone and 49.38 mg of potassium ferricyanide in 7.5 mL of ultrapure water, add 7.5 mL of 0.2 M hydrochloric acid and stir for 30 minutes. Place in a microwave reactor and react at 80℃ for 20 minutes. After cooling, centrifuge at 12000 rpm and 10℃ for 15 minutes in a low-temperature high-speed centrifuge to obtain a deep blue precipitate, Prussian blue (PB).

[0069] The properties of PB nanoparticles prepared by the above two methods were characterized, specifically by using a Hitachi H-7650 transmission electron microscope. Figure 1 The results show that, compared with the hydrothermal method, the PB nanoparticles prepared by the microwave synthesis method have more uniform morphology and better dispersibility; moreover, the PB prepared by the microwave synthesis method has a higher yield, indicating that this method is superior to the hydrothermal method in the preparation of PB. Figure 2 ).

[0070] (2) Preparation of mesoporous Prussian blue nanoparticles

[0071] At ambient temperature and pressure (15–35°C, 1 atm), 20 mg of Prussian blue and 100 mg of polyvinylpyrrolidone were dissolved in 20 mL of hydrochloric acid (2M). After stirring for 30 minutes, the mixture was transferred to a polytetrafluoroethylene (PTFE) reactor. The temperature was raised from room temperature to 140°C within 60 minutes and maintained at this temperature for 4 hours. After the temperature naturally cooled to room temperature, the PTFE reactor was ultrasonically cleaned for 10 minutes. Then, it was centrifuged at 12,000 rpm and 10°C for 15 minutes to remove the supernatant and retain the precipitate. Anhydrous ethanol was added, and the precipitate was ultrasonically cleaned to redisperse it in anhydrous ethanol. After centrifugation, this operation was repeated 3 times to obtain a deep blue precipitate of hollow mesoporous Prussian blue (HMPB).

[0072] (3) Preparation of hollow mesoporous Prussian blue loaded with rapamycin

[0073] Take 20 mg of hollow mesoporous Prussian blue prepared in step (2) and 2 mg of rapamycin (Rapa) and dissolve them in 4 mL of anhydrous ethanol. Stir magnetically for 24 hours and centrifuge at 12000 rpm and 10 degrees Celsius for 15 minutes to obtain a precipitate. Add anhydrous ethanol to redisperse the precipitate and repeat the centrifugation 3 times to obtain hollow mesoporous Prussian blue (RH) loaded with rapamycin.

[0074] (4) Preparation of Prussian blue-loaded rapamycin nanoparticles modified with cell-penetrating peptides.

[0075] 20 mg of activated cell-penetrating peptide (ACPP) and 20 mg of polyethylene glycol were dissolved in 10 mL of ultrapure water. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to a final concentration of 0.1 M. After magnetic stirring for 8 hours, the solution was dialyzed for 24 hours, with a molecular weight cutoff of 5000 Da. The ultrapure water in the dialysis tank was replaced every 8 hours to obtain a clear solution. 20 mg of rapamycin-loaded hollow mesoporous Prussian blue prepared in step (3) was dissolved in this clear solution. The solution was magnetically stirred for 8 hours and centrifuged at 12000 rpm at 10°C for 15 minutes. Anhydrous ethanol was added for reselection of the precipitate. After repeating the centrifugation three times, rapamycin-loaded Prussian blue targeted nanoparticles (RHPA) were obtained. The nanoparticles were stored at 4°C.

[0076] The properties of the HMPB and RHPA nanoparticles prepared above were characterized specifically by using a Hitachi H-7650 transmission electron microscope. Figure 3 The results showed that both hollow mesoporous Prussian blue (HMPB) and targeted modified hollow mesoporous Prussian blue-loaded rapamycin (RHPA) exhibited good dispersibility, uniform size, and both had a hollow mesoporous structure. The particle size and zeta potential of HMPB and RHPA aqueous solutions were characterized using Nano-ZS (Malvern Insruments Limited). Figure 4 The RHPA nanoparticles have a hydrated particle size of approximately 240 nm and possess a high surface potential, which is beneficial for cellular absorption. Comparison of the X-ray diffraction pattern data of PB with the standard card confirms the accurate preparation of Prussian blue nanoparticles. Furthermore, by comparing the X-ray diffraction patterns of HMPB and RH with the PB data, we found that the peak angles did not change significantly, indicating that loading with rapamycin does not alter the structure of Prussian blue. Figure 5 ).

[0077] Example 2

[0078] 1. Total antioxidant capacity test

[0079] (1) ① Weigh 54.87 mg of 2,2'-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and dissolve it in 20 mL of phosphate buffer to obtain a 5 mM ABTS stock solution. Add excess manganese dioxide powder to the solution, stir well with a glass rod, and let stand for 5 minutes. Filter the reacted ABTS solution using qualitative filter paper, repeating the process 3 times to finally obtain the ABTS free radical solution (ABTS· +The filtered liquid was stored at -20°C overnight and used the next day. The ABTS solution was diluted with phosphate buffer and its absorbance at 734 nm was measured using a microplate reader. Subsequent experiments could only proceed when the absorbance was 0.4 ± 0.02.

[0080] ② RHPA or Rapa was prepared at different concentrations and added to ABTS free radical solution to obtain RHPA solutions with final Prussian blue concentrations of 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL (the final concentration of Rapa was the same as RHPA). The solutions were then detected using a microplate reader. Finally, the absorbance values ​​at 734 nm were plotted after subtracting the RHPA or Rapa itself to evaluate the total antioxidant capacity of RHPA and Rapa.

[0081] The results are as follows Figure 6 As shown: From Figure 6 As shown in Figure A, the absorbance decreases with increasing RHPA concentration, indicating that the scavenging rate increases accordingly. However, in... Figure 6 In sample B, the absorbance remained essentially the same as the control group despite changes in Rapa concentration. These results indicate that the targeted-modified hollow mesoporous Prussian blue nanoparticles loaded with rapamycin (RHPA) effectively scavenge free radicals, and rapamycin does not affect the antioxidant capacity of Prussian blue.

[0082] 2. Electron spin resonance instrumentation for detecting RHPA's ability to scavenge hydroxyl radicals.

[0083] ① First, we used the Fenton reaction to generate hydroxyl radicals (·OH): A 1.8 mM (final concentration) ferrous sulfate solution was mixed with a 5 mM hydrogen peroxide solution and reacted for 10 minutes. RHPA and PB were diluted separately with ultrapure water and then added to the ferrous sulfate and hydrogen peroxide mixture to a final concentration of 20 μg / mL. After shaking and mixing, the mixture was allowed to stand for 30 minutes. After 30 minutes, 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was added to capture the remaining free radicals. The solution was aspirated using a 0.5 mm capillary tube, and the remaining free radicals were detected using an electron spin resonance (EPR) analyzer (using the Fenton reaction mixture (ferrous sulfate and hydrogen peroxide solution) and DMPO as controls). The EPR intensity indicates the ability of RHPA and PB to scavenge hydroxyl radicals.

[0084] ② Next, we react potassium superoxide (K2O) and 18-crown ether-6 to generate the anolyte anion (·O2). -): Mix 2 mg / mL (final concentration) potassium superoxide with 8 mg / mL 18-crown ether-6 dimethyl sulfoxide (DMSO) solution and react for 30 minutes. Dilute RHPA and PB separately with ultrapure water (final concentration 20 μg / mL), add to the potassium superoxide (K2O) and 18-crown ether-6 mixed solution, shake well and let stand for 30 minutes. Perform intensity testing on EPR according to the detection method in ① to determine the ability of RHPA and PB to scavenge superoxide anions.

[0085] The results are as follows Figure 7 As shown in Figure A, the peak height alone indicates that PB has the ability to scavenge hydroxyl radicals, and RHPA also has a similar ability. Targeted modification and rapamycin loading do not affect their ability to scavenge hydroxyl radicals. The same results can be obtained from... Figure 7 As shown in section B, both RHPA and PB can scavenge superoxide anions. The results indicate that the prepared RHPA effectively scavenge hydroxyl radicals and superoxide anions, demonstrating the successful implementation of the experimental protocol.

[0086] Example 3

[0087] 1. Detection of cell death activity in human neuroblastoma cells induced by the RHPA reverse glucose-oxygen deprivation model

[0088] ① First, evaluate the effects of PB, HMPB, Rapa, or RHPA on the cell viability of human neuroblastoma cells SH-SY5Y. SH-SY5Y cells in logarithmic growth phase (from the American Model Culture Bank) were used at a cell number of 2 × 10⁶. 4 SH-SY5Y cells were seeded at 100 μL / well in 96-well plates and allowed to adhere for 24 hours. PB, HMPB, Rapa, or RHPA were then added to the 96-well plates to achieve final concentrations of 0, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL for PB, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL for RHPA, and final concentrations of 0, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL for Rapa. After culturing for another 24 hours, the viability of SH-SY5Y cells was assessed using the Cell Counting Kit-8. Cell viability (%) = (OD0)0 450 Experimental group / OD 450 (Control group) × 100%.

[0089] ② Take SH-SY5Y cells in logarithmic growth phase at a density of 2×10⁻⁶ 4SH-SY5Y cells were seeded at a density of 1 / mL into 96-well plates and allowed to adhere for 24 hours. The 96-well plates then inoculated with SH-SY5Y cells were placed in a glucose-oxygen deprivation incubator (5% CO2, 0.1% Oxygen, 94.9% Nitrogen) for 6 hours. RHPA was then added to the 96-well plates to final concentrations of 0, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL. Simultaneously, the 96-well plates inoculated with SH-SY5Y cells were transferred back to normal culture conditions. After 24 hours, cell viability was assessed according to method ①.

[0090] Cell survival rate, such as Figure 8 As shown in Figure A, when the concentration of PB, HMPB, or RHPA was 20 μg / mL, the viability of SH-SY5Y cells could be maintained at around 80%, and concentrations below 5 μg / mL could maintain cell viability at around 90%. These results indicate that the prepared PB, HMPB, or RHPA at concentrations below 5 μg / mL had no significant effect on the growth of SH-SY5Y cells. However... Figure 8 As shown in Figure B, when the Rapa concentration was 10 μg / mL, cell viability was 50%, decreasing to about 10% at 20 μg / mL, indicating that Rapa itself has a certain inhibitory effect on SH-SY5Y cell growth. Figure 8 In C, the survival rate of SH-SY5Y cells treated with the glucose-oxygen deprivation model alone was 57.5%; the survival rate of SH-SY5Y cells induced by RHPA at a concentration of 2.5 μg / mL was 87.9%. The results show that the prepared RHPA can reverse the cell death of human neuroblastoma cells induced by the glucose-oxygen deprivation model, proving that RHPA can effectively inhibit neuronal death caused by oxidative stress, indicating that the experimental protocol was successfully implemented.

[0091] 2. Detection of apoptosis activity in human neuroblastoma cells induced by the RHPA reverse glucose-oxygen deprivation model

[0092] Apoptosis and mitochondrial damage are the main mechanisms of cell death induced by the glucose-oxygen deprivation model. To further investigate the potential mechanisms by which the RHPA-reversed glucose-oxygen deprivation model induced cell death in human neuroblastoma cells, we used flow cytometry to detect and analyze mitochondrial membrane potential and apoptosis in each treatment group. The specific methods are as follows:

[0093] (1) First, SH-SY5Y human neuroblastoma cells in the logarithmic growth phase were selected at a density of 2×10⁻⁶. 4Cells per 6 mL (6 mL) were seeded in 6 cm culture dishes and allowed to adhere for 24 hours. The cells were divided into three groups: a blank control group, a glucose-oxygen deprivation model group, and a glucose-oxygen deprivation model + 1 μg / mL RHPA group. The blank control group was supplemented with the same amount of PBS buffer as the drug. After 24 hours of cell adhesion, the culture dishes except the blank control group were transferred to a glucose-oxygen deprivation model incubator (5% CO2, 0.1% Oxygen, 94.9% Nitrogen) and cultured for 6 hours. Then, 1 μg / mL RHPA was added, and the culture dishes were transferred back to normal culture conditions. Cells in each group were then incubated for another 24 hours. After incubation, the old culture medium was collected from the culture dishes, washed once with PBS, and the washed PBS was also collected. Then, 1 mL of 0.25% (w / v) trypsin digestion solution was added, and digestion was carried out for 2 minutes. The recovered culture medium was added to the digestion solution to stop digestion. All cells were then collected into 15 mL centrifuge tubes, and any remaining cells in the culture dishes were washed with PBS and collected into the corresponding centrifuge tubes. Centrifuge the cells in the centrifuge tubes at 1500 rpm for 10 minutes on a horizontal centrifuge, remove the supernatant, resuspend the cells in 2 mL of 0.09% NaN3 (w / v) phosphate buffer, and count the cells using a cell counter.

[0094] (2) Based on the cell concentration obtained after cell counting, 1×10⁻⁶ cells from different groups were taken from the solution. 6 For each cell, bring the volume to 1 mL with 0.09% (w / v) NaN3 phosphate buffer. Add 1 μL of mitochondrial membrane potential fluorescent probe JC-1 to each tube and incubate at 37°C for 15–30 minutes in a 5% CO2 incubator. After incubation, centrifuge at 1500 rpm for 10 minutes using a horizontal centrifuge, remove the supernatant, add 500 μL of phosphate buffer, and gently resuspend the cells in the solution using a pipette. Then, analyze these cell samples using a Beckman flow cytometer. Before processing, filter the cells through a 300-mesh (40–50 μm pore size) nylon mesh, with at least 10,000 cells analyzed for each sample.

[0095] (3) Based on the cell concentration obtained after cell counting, 1×10⁻⁶ cells from different groups were taken from the solution. 6For each cell, bring the volume to 500 μL using 0.09% (w / v) NaN3 phosphate buffer. Add 5 μL Annexin-V and 10 μL PI to each tube and incubate at 37°C for 15–30 minutes in a 5% CO2 incubator. After incubation, add 500 μL of 0.09% (w / v) NaN3 phosphate buffer to each cell tube. Then, analyze these cell samples using a Beckman flow cytometer. Before processing, filter the cells through a 300-mesh (40–50 μm pore size) nylon mesh. On the flow cytometer, Annexin V-FITC (Ex = 488 nm; Em = 530 nm) is detected through the FITC detection channel and PI is detected through the PI detection channel (Ex = 535 nm; Em = 615 nm).

[0096] Experimental results are as follows Figure 9 As shown, in Figure 9 In A, we found that the apoptosis rate in SH-SY5Y cells treated with the glucose-oxygen deprivation model was as high as 32.5%. After incubation with 1 μg / mL RHPA for 24 hours, the apoptosis rate in SH-SY5Y cells significantly decreased to 13.6%, indicating that RHPA can effectively reverse apoptosis induced by the glucose-oxygen deprivation model. Meanwhile, in Figure 9 In sample B, the proportion of mitochondrial membrane potential flipping in SH-SY5Y cells treated with the glucose-oxygen deprivation model was as high as 30.3%. After incubation with 1 μg / mL RHPA for 24 hours, the number of mitochondrial membrane potential flipping in SH-SY5Y cells significantly decreased, returning to approximately normal levels. This indicates that RHPA can effectively reverse the membrane potential flipping caused by mitochondrial damage induced by the glucose-oxygen deprivation model. These results demonstrate that the prepared RHPA can reverse the effects of glucose-oxygen deprivation model on mitochondrial membrane potential flipping and apoptosis in human neuroblastoma cells, indicating the successful implementation of the experimental protocol.

[0097] 3. Therapeutic effect of RHPA in a mouse model of spinal cord injury

[0098] A mouse model of thoracic spinal cord impingement was used to evaluate the therapeutic effect of RHPA on spinal cord injury. We used a spinal cord impingement device (NYUImpactorModelII) to impinge C57 / B6J mice (23–25 g, female, purchased from Guangdong Provincial Medical Laboratory Animal Center) to create a spinal cord injury model. The specific procedures were as follows: all surgical instruments were sterilized at high temperature, and the mice were intraperitoneally anesthetized with a tribromoethanol mixture (13 uL / g). The dorsal skin and muscles were incised to expose the thoracic spinous processes. Under a microscope, the T12 lamina was carefully dissected and lifted to fully expose the spinal cord, forming a square window approximately 2.0 mm in diameter. The mice were then fixed on the spinal cord impingement device (NYUImpactorModelII), and a 10 g impact at a height of 6.5 mm was applied to create a spinal cord heavy-weight impingement injury model at the exposed spinal cord site. After hemostasis, the deep fascia, subcutaneous tissue, and skin were sutured sequentially. The experimental groups were divided into: sham-operated group (Sham), saline group (SCI), HMPB (10 mg / kg), Rapa (1 mg / kg), RH (10 mg / kg), and RHPA (10 mg / kg) group. Immediately after spinal cord injury modeling, the aforementioned nanomedicines were injected into mice via tail vein injection, and the mice were simultaneously incubated at 37°C until they naturally awakened. Behavioral scores were assessed at different time points (1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, and 4 weeks). At 4 weeks, rotarod testing, Nissl staining, and immunofluorescence staining were performed.

[0099] Behavioral scoring results as follows Figure 10 As shown, we evaluated the six groups of mice according to the BassoMouseScale (BMS) behavioral scoring system (doi:10.1089 / neu.2006.23.635; JNeurotrauma.2006.23(5):635-59.). All mice in the Sham group exhibited normal behavior, scoring 9 points. Compared to other groups, the RHPA group showed significant improvement starting at week 2, reaching an average score of around 5 points by week 4.

[0100] Three days prior to the experiment, mice were trained to use the equipment. They were placed on an accelerator bar, starting at 5 rpm and accelerating to 80 rpm within 5 minutes. The time it took for each mouse to fall off the bar was recorded. Each mouse was tested twice, and the average time was recorded. The results of the rotundus experiment are as follows: Figure 11 As shown, we conducted a rotarod experiment on 6 groups of mice at week 4 after injury. It can be seen that at week 4 after injury, the mice in the saline group fell off the rotarod in about 20.5 seconds, while after RHPA treatment, this time increased to 47.3 seconds. The above results indicate that RHPA has the effect of promoting the recovery of motor function and hind limb motor coordination after spinal cord injury in mice.

[0101] Nissl staining began with stepwise dewaxing of the tissue to water. The sections were then immersed in 0.1% tar violet working solution, heated in a 50°C oven for 30 minutes, washed with distilled water for 5 minutes, differentiated with Nissl differentiation solution for 5-30 seconds, washed with distilled water for 5 minutes, and dewaxed and cleared using a gradient of alcohol to xylene. The sections were then mounted with neutral resin. Five stained sections were randomly selected from each mouse, and the average number of surviving neurons in the anterior horn of the spinal cord was calculated. Results are as follows: Figure 12 As shown, we performed spinal cord sections on mice in each group 4 weeks after injury. We were able to observe Nissl staining of anterior horn motor neurons in the spinal cord. The number of anterior horn motor neurons in the spinal cord injury group was significantly reduced compared to the sham-operated group. However, in the RHPA group, the number of surviving anterior horn motor neurons was significantly increased, indicating that RHPA can promote motor neuron survival.

[0102] Immunofluorescence staining: Preparation of frozen sections of spinal cord tissue: A complete 1cm section of spinal cord tissue from the damaged area was harvested, fixed, dehydrated, embedded, and sectioned. The sections were attached to glass slides and baked for 2 hours. Staining: The slides were washed three times with PBS for 5 minutes each time; the sections were placed in PBS and microwaved on medium heat for 10 minutes for repair; washed three times with PBS for 5 minutes each time, then immersed in 0.3% PBST solution and perforated for 15 minutes; then blocked with 5% goat serum and 3% BSA at room temperature for 1 hour; diluted primary antibody was added, and incubated overnight at 4°C. The next day, the slides were washed three times with PBS for 5 minutes each time; secondary antibody was added, and incubated at room temperature in the dark for 2 hours; washed three times with PBS for 5 minutes each time; finally, the slides were mounted with a DAPI-containing anti-fluorescence quencher. Immunofluorescence results are as follows. Figure 13 As shown, the proportion of pro-inflammatory M1 macrophages in the spinal cord injury site was significantly increased in the spinal cord injury group compared to the sham surgery group. After RHPA treatment, the proportion of M1 macrophages decreased significantly. The results indicate that RHPA exerts an anti-inflammatory effect at the injury site.

[0103] Scavenging free radicals and reducing inflammatory responses are important issues in the treatment of spinal cord injury (SCI). To address this, this invention designs hollow mesoporous Prussian blue nanoparticles as drug carriers to load the anti-inflammatory drug rapamycin. A major challenge in administering rapamycin to animals with central nervous system injury is the blood-brain barrier / blood-spinal cord barrier, which hinders drug entry into the central nervous system. Based on the characteristics of transient opening of the blood-spinal cord barrier and the high expression of matrix metalloproteinases (MMPs) at the site of spinal cord injury, we modified the surface of Prussian blue with MMP-responsive, cell-penetrating peptides, ultimately constructing a hollow mesoporous Prussian blue nanosystem (RHPA) modified with cell-penetrating peptides to support rapamycin.

[0104] RHPA exhibits strong enzyme-mimicking ability in vitro by scavenging ROS, protecting human neuroblastoma cells from oxygen-glucose deprivation-induced apoptosis. Simultaneously, RHPA demonstrates in vivo effects in promoting motor function recovery and increasing nerve survival. These results indicate that RHPA has effective neuroprotective effects in a mouse model of spinal cord injury, reducing apoptosis and promoting motor function recovery after injury.

[0105] In summary, this invention not only demonstrates the preparation of a hollow mesoporous Prussian blue nanosystem modified with cell-penetrating peptides that can activate cells, but also provides more evidence for the neuroprotective mechanism of Prussian blue nanomaterials and their future clinical application in the treatment of spinal cord injury.

[0106] The above examples illustrate different implementation processes of the present invention in detail. However, the implementation methods of the present invention are not limited thereto. Those skilled in the art can achieve the purpose of the present invention based on the content disclosed in the present invention. Any improvements and modifications made based on the concept of the present invention fall within the protection scope of the present invention. The specific protection scope is subject to the claims.

Claims

1. A method for preparing Prussian blue-loaded rapamycin-based targeted nanoparticles, characterized in that, Includes the following steps: (1) Preparation of Prussian blue nanoparticles; (2) Preparation of hollow mesoporous Prussian blue nanoparticles; (3) Preparation of hollow mesoporous Prussian blue loaded with rapamycin: Dissolve rapamycin in anhydrous ethanol, add it to the anhydrous ethanol solution of hollow mesoporous Prussian blue nanoparticles, stir and centrifuge, retain the precipitate, and obtain hollow mesoporous Prussian blue loaded with rapamycin. (4) Preparation of rapamycin-loaded Prussian blue targeted nanoparticles: Polyethylene glycol and cell-penetrating peptides were dissolved in ultrapure water, and N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and stirred. Then, dialysis was performed. Hollow mesoporous Prussian blue loaded with rapamycin prepared in step (3) was added to the dialysis solution, stirred, and centrifuged to obtain rapamycin-loaded Prussian blue targeted nanoparticles.

2. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 1, characterized in that, In step (3), the mass-to-volume ratio of rapamycin to anhydrous ethanol is 0.1–1 mg / mL; the mass-to-volume ratio of hollow mesoporous Prussian blue nanoparticles to anhydrous ethanol is 1–10 mg / mL.

3. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 2, characterized in that, In step (4), the mass-to-volume ratio of polyethylene glycol to ultrapure water is 0.1–1 mg / mL; the mass-to-volume ratio of cell-penetrating peptides to ultrapure water is 0.1–1 / mL.

4. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 3, characterized in that, The amounts of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride used in step (4) are calculated at 0.1 mol per milliliter (mL).

5. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to any one of claims 1-4, characterized in that, Step (1) Prussian blue nanoparticles can be prepared by microwave synthesis or hydrothermal method.

6. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 5, characterized in that, The Prussian blue nanoparticles were prepared by microwave synthesis as follows: potassium hexacyanoferrate(II) trihydrate and polyvinylpyrrolidone were weighed and dissolved in ultrapure water, dilute hydrochloric acid solution was added, and the mixture was stirred until a clear solution was obtained. The reaction was carried out in a microwave reactor, centrifuged and resuspended, and dried to obtain Prussian blue nanoparticles.

7. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 6, characterized in that, The mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 5-7:1 mg / mL; the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.1-0.3 g / mL; the concentration of dilute hydrochloric acid is 0.05-0.4 mol / L; and the reaction temperature in the microwave reactor is 70-90℃, and the reaction time is 10-40 min.

8. The method for preparing rapamycin-loaded Prussian blue targeted nanoparticles according to claim 6 or 7, characterized in that, The specific process for preparing hollow mesoporous Prussian blue nanoparticles in step (2) is as follows: Polyvinylpyrrolidone and the Prussian blue nanoparticles prepared in step (1) are dissolved in hydrochloric acid, and then the reaction is carried out in a polytetrafluoroethylene reactor. After the reaction is completed, the temperature is lowered and centrifuged to obtain a dark blue precipitate, which is the hollow mesoporous Prussian blue nanoparticle.

9. A rapamycin-loaded Prussian blue-targeting nanoparticle, prepared by any one of the methods of claims 1-8.

10. The use of the rapamycin-loaded Prussian blue targeted nanoparticles of claim 9 in the preparation of a medicament for treating spinal cord injury.

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