Magnetic drug-loaded liposome for nerve repair and preparation method thereof

Abstract

This invention discloses a magnetic drug-loaded liposome for nerve repair and its preparation method. The magnetic drug-loaded liposome prepared by this method contains magnetic nanoparticles and a drug. The mass fractions of egg yolk lecithin, cholesterol, DSPE-MPEG 2000, BFe3O4, and the drug in 1 mL of the liposome solution are 0.5%–1.5%, 0.15%–0.35%, 0.1%–0.35%, 0.1%–0.2%, and 0.1%–0.2%, respectively. The drug is one or more of the natural antioxidants such as piracetamine, resveratrol, or tea polyphenols. The multifunctional magnetic drug-loaded liposome provided by this invention, combined with magnetic field loading, can be used for the repair of damaged nerve cells.

Description

Technical Field

[0001] This invention relates to a method for preparing liposomes, and more specifically, to a magnetic drug-loaded liposome for nerve repair and a method for preparing the same. Background Technology

[0002] Ischemic stroke is a clinical syndrome caused by narrowing or occlusion of the brain's blood supply arteries (such as the carotid and vertebral arteries), resulting in insufficient blood supply to the brain and leading to localized or widespread ischemia and hypoxic necrosis of brain tissue, ultimately resulting in a series of neurological deficits. Ischemic stroke has a high incidence and mortality rate, and is recognized worldwide as one of the most serious public health problems. Ischemia triggers an ischemic cascade, involving a series of biochemical reactions, including energy depletion, ion imbalance and excitotoxicity, oxidative stress, cell death (apoptosis or necrosis), activation of the complement system, and the initiation of inflammatory and immune responses, ultimately leading to irreversible damage. The clinical treatment for ischemic stroke involves thrombolysis using tissue plasminogen activator to restore blood flow. However, the time window for thrombolysis is relatively narrow, so only a small number of patients can receive timely thrombolytic therapy and benefit from it. Furthermore, neurons in the central nervous system are inherently non-regenerative and non-proliferating; therefore, how to repair neurons and block neuronal apoptosis after nerve injury has undoubtedly become a hot research topic in neuroscience.

[0003] Liposomes, in the form of closed phospholipid bilayer spheres at the nanoscale, are widely used as carriers for encapsulating genes, drugs, and contrast agents due to their excellent biocompatibility, for the diagnosis and treatment of central nervous system diseases. Liposomes possess a unique amphiphilic structure, capable of encapsulating both hydrophilic and hydrophobic compounds. Their core space is hydrophilic, allowing them to encapsulate hydrophilic drugs and nucleic acids, delivering the encapsulated material to the target disease area and providing a degree of sustained drug release. Conversely, their phospholipid bilayer is hydrophobic, allowing them to encapsulate lipophilic compounds. However, in practical applications, nanoliposomes are susceptible to phagocytosis or adsorption by other substances in the bloodstream. To address this issue, researchers have modified their surfaces with various polymers, targeting molecules, or aptamers, further improving their efficiency in tissue delivery.

[0004] Taxifolin, also known as dihydroquercetin, is a bioflavonoid extracted from the roots of larch trees in high-altitude regions. It is a natural and potent antioxidant. Studies have shown that taxifolin has outstanding medicinal value in antioxidation, anti-inflammation, liver protection, anti-tumor activity, lipid regulation, anti-angiogenesis, anti-Alzheimer's disease, and antibacterial properties. In addition, it can prevent liver damage caused by carbon tetrachloride and inhibit erythrocyte lysis caused by oxidation. As an excellent anti-inflammatory substance, it can effectively reduce the expression of interleukin-6 and interleukin-8 in tissues.

[0005] Tea polyphenols are compounds widely found in tea leaves. They are believed to possess various physiological activities, including antioxidant, anti-inflammatory, anti-cancer, blood pressure-lowering, blood sugar-lowering, and anti-aging effects. These activities help prevent various diseases and are beneficial to the cardiovascular, immune, and metabolic systems. Their antioxidant activity is 2.6 times that of butylated hydroxyanisole (BHA) and 3.6 times that of vitamin E.

[0006] Resveratrol, primarily derived from plants such as peanuts, grapes (red wine), Japanese knotweed, and mulberries, is a natural polyphenol compound and a natural antioxidant. Studies have shown that resveratrol possesses physiological activities including antioxidant, anti-inflammatory, anti-cancer, lipid-lowering, blood sugar-lowering, and cardiovascular health-promoting effects. In terms of neuroprotection, it can scavenge free radicals in the body, reduce oxidative stress damage to nerve cells, and promote nerve cell growth and regeneration. It exerts its neuroprotective effect by regulating multiple signaling pathways, such as the signal transduction of nerve growth factor. Summary of the Invention

[0007] One objective of this invention is to provide a magnetic drug-loaded liposome with multiple functions, including antioxidant, anti-apoptotic, and nerve regeneration promotion. The liposome has a phospholipid bilayer structure, a surface modified with PEG (polyethylene glycol), and encapsulates magnetic nanoparticles and drugs. Furthermore, it combines the excellent magnetic properties of the magnetic nanoparticles with the strong antioxidant properties of the drugs. The mass fractions of egg yolk lecithin, cholesterol, DSPE-MPEG2000, BFe3O4, and drugs in 1 mL of the liposome solution are 0.5%–1.5%, 0.15%–0.35%, 0.15%–0.35%, 0.1%–0.2%, and 0.1%–0.2%, respectively.

[0008] The aforementioned drug is a natural antioxidant used for nerve repair. Specifically, the drug is one or more of the following: piperidine, resveratrol, or tea polyphenols.

[0009] The liposomes of this invention contain therapeutic drugs for nerve injury, which are natural and potent antioxidants with great potential for neuroprotection after injury. Furthermore, by encapsulating the drug in liposomes, this invention effectively improves drug bioavailability, reduces treatment frequency, and enhances patient compliance. The liposomes of this invention contain magnetic nanoparticles as therapeutic agents. Combined with magnetic field loading, they can promote nerve repair through magnetobiological effects. Simultaneously, guided by an external magnetic field, the magnetically loaded piracetamine liposomes can be directionally delivered to the damaged nerve area, increasing the local drug concentration, thereby enhancing the therapeutic effect and reducing the impact on healthy tissue. The combination of magnetic properties, potent antioxidant capabilities, and neuroprotective functions in liposomes represents an innovative therapeutic strategy.

[0010] The second objective of this invention is to provide a method for preparing multifunctional magnetic drug-loaded liposomes for nerve repair, comprising the following steps:

[0011] The target components designed were: egg yolk lecithin, cholesterol, DSPE-MPEG2000, BFe3O4, and the drug, with mass fractions of 0.5%–1.5%, 0.15%–0.35%, 0.15%–0.35%, 0.1%–0.2%, and 0.1%–0.2%, respectively, in 1 mL of liposome solution.

[0012] Step 1: Prepare Fe3O4 magnetic nanoparticles using a co-precipitation method;

[0013] FeCl2·4H2O and FeCl3·6H2O were added to deionized water and magnetically stirred at a water bath temperature of 70℃~85℃ for 0.5h. Then ammonium hydroxide was added and stirring was continued for 3h~4h. The product was then removed, washed twice with deionized water and twice with ethanol. After washing, the product was dried in a freeze dryer at -30℃~-50℃ for 18h~24h to obtain Fe3O4 magnetic nanoparticles.

[0014] Dosage: 1g-3g of FeCl2·4H2O, 2g-4g of FeCl3·6H2O, and 10mL-20mL of ammonium hydroxide are required for 100mL of deionized water;

[0015] The ammonium hydroxide has a mass percentage concentration of 25%.

[0016] The magnetic stirrer can be adjusted from 400 rpm to 1000 rpm.

[0017] Step 2: Modification with magnetic nanoparticles;

[0018] Bovine serum albumin was added to a Fe3O4 magnetic nanoparticle solution and stirred vigorously at 0°C for 10 h to obtain a BFe3O4 solution.

[0019] Dosage: Add 30mg to 80mg of bovine serum albumin to 10mg of Fe3O4 magnetic nanoparticles.

[0020] The magnetic stirrer can be adjusted from 800 rpm to 1200 rpm.

[0021] To prevent particle aggregation and ensure stable dispersion of Fe3O4 magnetic nanoparticles, bovine serum albumin (BSA) was used to modify the Fe3O4 magnetic nanoparticles.

[0022] Step 3: Prepare lipid films by rotary evaporation under reduced pressure;

[0023] Egg yolk lecithin, cholesterol, DSPE-PEG2000 and drugs were dissolved in an ethanol solution and ultrasonically vortexed for 2 minutes. The mixture was then transferred to a round-bottom flask of a rotary evaporator and the ethanol was removed by rotary evaporation under reduced pressure at 50°C and 50-100 rpm. Finally, a uniform lipid film was formed at the bottom of the round-bottom flask.

[0024] The drug is a natural antioxidant used for nerve repair. Specifically, the drug is one or more of the following: piperidine, resveratrol, or tea polyphenols.

[0025] Dosage: 30mg-90mg of egg yolk lecithin, 5mg-15mg of cholesterol, 5mg-15mg of DSPE-PEG2000, and 3mg-9mg of the drug are required in 50mL of ethanol;

[0026] The ultrasonic frequency is 60kHz.

[0027] DSPE-PEG2000 refers to distearylphosphatidylethanolamine-polyethylene glycol 2000.

[0028] Step 4: Liposomes are obtained after hydration;

[0029] Add BFe3O4 solution to the rotary evaporator for hydration, and sonicate until the lipid film at the bottom of the rotary evaporator is completely detached; then place it in a water bath at 30℃~60℃ and stir continuously, and use a pipette to blow the rotary evaporator to collect the lipid suspension; transfer the collected suspension to a sample bottle, and sonicate it at 0℃ using the probe of a cell disruptor for 3min~10min, i.e., sonicate for 3s, stop for 2s, and finally filter the product to obtain a clear and homogeneous magnetic drug-loaded liposome solution.

[0030] The ultrasonic frequency is 60kHz.

[0031] The magnetic stirrer can be adjusted from 350 rpm to 600 rpm.

[0032] The power of the cell disruptor is 150W to 300W.

[0033] Compared with the prior art, the main advantages of this invention are:

[0034] 1. The liposomes of this invention contain therapeutic drugs for nerve injury, which are natural and potent antioxidants with great potential for neuroprotection after injury. However, when the drugs are used directly, their bioavailability is low, limiting their efficacy. This invention, by encapsulating the drug in liposomes, effectively improves the drug's bioavailability. Simultaneously, the liposomes possess excellent controlled-release properties, allowing for sustained drug release and increasing the drug's circulation time in the body, thereby achieving better therapeutic effects, reducing treatment frequency, and improving patient compliance.

[0035] 2. The liposomes of the present invention contain magnetic nanoparticles as therapeutic agents. Combined with the loading of a magnetic field, they can promote nerve repair through magnetobiological effects. At the same time, guided by an external magnetic field, the magnetic drug-loaded liposomes can be directionally delivered to the damaged nerve area, increasing the local concentration of the drug, thereby enhancing the therapeutic effect and reducing the impact on healthy tissues.

[0036] 3. In addition to being used for guided delivery and as a therapeutic agent, the magnetic properties of magnetic drug-loaded liposomes can also be applied to fields such as magnetic resonance imaging to monitor treatment effects, thus possessing multiple functions.

[0037] 4. Combining magnetic properties with the potent antioxidant properties of piperidine and neuroprotective functions, liposomes represent an innovative treatment strategy that fully leverages the advantages of nanotechnology, magnetic guidance, and repair, providing a new therapeutic approach for nerve injury repair. Attached Figure Description

[0038] Figure 1 This is a structural diagram of magnetic liposomes loaded with taurine.

[0039] Figure 2 This is a transmission electron microscope image of Fe3O4 nanoparticles.

[0040] Figure 3 This is a transmission electron microscope image of taxonomic liposomes (Tax-Lipo).

[0041] Figure 4 This is a transmission electron microscope image of magnetic liposomes (Tax-BFe3O4-Lipo) loaded with taurine.

[0042] Figure 5 These are the XRD patterns of Tax-Lipo, BFe3O4, and Tax-BFe3O4-Lipo.

[0043] Figure 6 This is the hysteresis curve of Fe3O4.

[0044] Figure 7 These are the hysteresis curves of BFe3O4 and Tax-BFe3O4-Lipo.

[0045] Figure 8 These are the Zeta potential results for Lipo, Tax-Lipo, BFe3O4, and Tax-BFe3O4-Lipo.

[0046] Figure 9 This is the standard curve for piperidine solution.

[0047] Figure 10These are in vitro drug release curves for an ethanol solution (Tax) containing taxine and magnetic liposomes loaded with taxine (Tax-BFe3O4-Lipo).

[0048] Figure 11 These are bright-field images of normally cultured neurons.

[0049] Figure 12 These are bright-field images of neurons 4 hours after OGD injury.

[0050] Figure 13 The study investigated the effect of using a magnetic field in combination with taurine liposomes and taurine-loaded magnetic liposomes on neuronal survival.

[0051] Figure 14 The results are TUNEL staining of cells after treatment with a magnetic field using a combination of piperidine liposomes and magnetic liposomes loaded with piperidine.

[0052] Figure 15 This is the statistical result of cell apoptosis after treatment with a magnetic field by combining piperidine liposomes and magnetic liposomes loaded with piperidine.

[0053] Figure 16 This is the Real-Time PCR result of BAX in hypoxic nerve cells under the combined action of a magnetic field and liposomes containing taurine and magnetic liposomes loaded with taurine.

[0054] Figure 17 This is the Real-Time PCR result of Caspase 3 in hypoxic nerve cells under the combined action of a magnetic field and liposomes loaded with taurine.

[0055] Figure 18 This is the Real-Time PCR result of BCL2 in hypoxic nerve cells under the combined action of a magnetic field and liposomes loaded with taurine.

[0056] Figure 19 This is the Real-Time PCR result of NGF detection in hypoxic neurons under the combined action of a magnetic field on piracetamine liposomes and piracetamine-loaded magnetic liposomes.

[0057] Figure 20 This is the Real-Time PCR result of BDNF in hypoxic neurons under the combined action of a magnetic field and liposomes loaded with taurine. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The examples of the parameters listed are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0059] Example 1: Magnetic Taxodiacetic Liposomes

[0060] (I) Synthesis of magnetically loaded piperine liposomes

[0061] The specific synthesis process of magnetically loaded tamariscin liposomes is as follows:

[0062] Step 1: Prepare Fe3O4 magnetic nanoparticles using a co-precipitation method;

[0063] Add 1.07 g of FeCl2·4H2O and 2.91 g of FeCl3·6H2O to 100 mL of deionized water; after magnetic stirring for 0.5 h at a water bath temperature of 80 °C, add 20 mL of ammonium hydroxide and continue magnetic stirring for 3 h to obtain the first mixture.

[0064] The mixture was washed twice with deionized water and then twice with ethanol to obtain the washed product.

[0065] The washed product was placed in a freeze dryer at -50°C and dried for 24 hours. The product was then removed to obtain Fe3O4 magnetic nanoparticles.

[0066] The magnetic stirring speed is 800 rpm.

[0067] The mass percentage concentration of ammonium hydroxide is 25%.

[0068] Step 2: Modification with magnetic nanoparticles;

[0069] Add 10 mg of Fe3O4 magnetic nanoparticles to 100 mL of deionized water and mix well to obtain a second mixed solution.

[0070] 80 mg of bovine serum albumin (BSA) was added to the second mixed solution, and the mixture was stirred vigorously at 0 °C for 10 hours to obtain a BFe3O4 solution.

[0071] The dosage is as follows: the mass ratio of bovine serum albumin to Fe3O4 magnetic nanoparticles is 8:1.

[0072] In this invention, to prevent particle aggregation and ensure stable dispersion of Fe3O4 magnetic nanoparticles, bovine serum albumin is used to modify the magnetic nanoparticles.

[0073] Step 3: Prepare lipid films by rotary evaporation under reduced pressure;

[0074] 40 mg of egg yolk lecithin, 10 mg of cholesterol, 10 mg of DSPE-MPEG 2000, and 4 mg of taurine (Tax) were dissolved in 50 mL of ethanol solution and mixed by ultrasonic vortexing for 2 min to obtain a third mixed solution. The mixed solution was then transferred to a 250 mL round-bottom flask of a rotary evaporator and the ethanol was removed by rotary evaporation under reduced pressure at 50 °C and 100 rpm, finally forming a uniform lipid film at the bottom of the rotary evaporator flask.

[0075] The abbreviation for distearylphosphatidylethanolamine-polyethylene glycol 2000 is DSPE-MPEG 2000.

[0076] Step 4: Liposomes are obtained after hydration;

[0077] 4 mL of BFe3O4 solution was added to the rotary evaporator for hydration, and the mixture was sonicated until the lipid film at the bottom of the evaporator was completely detached. The flask was then incubated in a 50°C water bath with continuous stirring, and the lipid suspension was collected by pipetting with a pipette. The collected suspension was transferred to a 10 mL sample vial and intermittently sonicated for 10 min using a cell disruptor probe under ice conditions. Finally, the product was filtered to obtain a clear and homogeneous magnetic piperidine-loaded liposome solution.

[0078] The ultrasonic power is 150W. Intermittent ultrasonic treatment refers to cyclic ultrasound with 3 seconds of ultrasound followed by 2 seconds of pause.

[0079] The structural diagram of the magnetic piperidine-loaded liposomes prepared by the method in Example 1 is shown below. Figure 1 As shown in the figure, piperidin and BFe3O4 are encapsulated inside liposomes.

[0080] (II) Characterization of magnetic piperine-carrying liposomes

[0081] (A) Transmission electron microscopy (TEM) analysis: The morphology of Fe3O4 nanoparticles, taxine liposomes (Tax-Lipo), and magnetic liposomes loaded with taxine (Tax-BFe3O4-Lipo) were observed under a transmission electron microscope, as follows: Figure 2 , Figure 3 , Figure 4 As shown in the figure. An appropriate amount of the liposome solution to be tested was taken and diluted to a suitable concentration with PBS buffer. The solution was then dropped onto a copper grid covered with a carbon film and allowed to dry at room temperature. Phosphotungstic acid solution was used as a negative staining background, and the results were observed and photographed using TEM. The results showed that the liposomes loaded with taxine (Tax-Lipo) and the magnetic drug-loaded liposomes (Tax-BFe3O4-Lipo) both exhibited complete spherical shapes with particle sizes between 100 and 200 nm.

[0082] (B) X-ray diffraction (XRD) analysis: X-ray diffraction analysis was performed on different samples using an X-ray diffractometer. A Co target was scanned within the range of 10° to 90° at a scan rate of 5° / min. Figure 5 As shown. XRD results show that the synthesized Fe3O4, BFe3O4, and Tax-BFe3O4-Lipo materials all have six distinct diffraction peaks, corresponding to the (311), (440), (422), (440), (531), and (533) crystal planes of Fe3O4, respectively. The characteristic peaks are relatively sharp, reflecting their good crystallinity. After modifying BSA with Fe3O4 and adding it to liposomes, although the characteristic peaks of Fe3O4 crystals were slightly weakened, no impurity peaks appeared, indicating that the structure of Fe3O4 was completely preserved in this material.

[0083] (C) Vibrating Sample Magnetometer (VSM) Analysis: Hysteresis loops of Fe3O4, BFe3O4, and Tax-BFe3O4-Lipo samples were obtained using a vibrating sample magnetometer, such as... Figure 6 , Figure 7 As shown in the figure. The results indicate that the curve passes through the origin, reflecting the absence of remanence in the material when the external magnetic field is removed, suggesting that the synthesized Fe3O4, BFe3O4, and Tax-BFe3O4-Lipoo possess superparamagnetism. Specifically, the maximum specific saturation magnetization of Fe3O4 is 60.02 emu / g, that of BFe3O4 is 4.94 emu / g, and that of Tax-BFe3O4-Lipoo is 2.10 emu / g.

[0084] (D) Zeta potential measurement: The zeta potential of BSA-modified Fe3O4 magnetic nanoparticles (BFe3O4), liposomes (Lipo), tataric acid-loaded liposomes (Tax-Lipo), and tataric acid-loaded magnetic liposomes (Tax-BFe3O4-Lipo) was measured using a laser particle size analyzer. All liposome samples were diluted 10-fold before detection. Detection was performed at room temperature (22℃), and all measurements were repeated three times. The final result was the average value. Figure 8The results show that the zeta potentials of BFe3O4, Lipo, Tax-Lipo, and Tax-BFe3O4-Lipo are all negative, at -12.7 mV, -45.6 mV, -26.4 mV, and -30.6 mV, respectively. For zeta potentials, higher values ​​(positive or negative) indicate stronger electrostatic repulsion and better stability of the lipid nanoparticles. However, higher zeta potentials may also increase the toxicity and immunogenicity of the lipid nanoparticles. Generally, an absolute zeta potential greater than ±30 mV is considered sufficiently stable, as it indicates that the particle surface carries sufficient charge to prevent aggregation between particles. Therefore, considering the balance between stability and safety, the designed Tax-BFe3O4-Lipo exhibits good stability, low toxicity, and low immunogenicity.

[0085] (E) Liposome Encapsulation Efficiency and Drug Loading Determination: The encapsulation efficiency was determined using low-speed centrifugation. The encapsulation efficiency of liposomes loaded with taxine was 93.3%, and the drug loading was 4.15%. The encapsulation efficiency of magnetic liposomes loaded with taxine was 77.4%, and the drug loading was 2.34%. To determine the total encapsulation volume of taxine liposomes, the liposomes, after being sonicated, were centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. 5.00 mL of ethanol was used to dissolve the precipitate, with ethanol as a reference. The solution was then added to a 96-well plate (200 μL well). The absorbance of the liposome solution at 290 nm was measured using a multi-functional microplate reader, based on the UV absorption spectrum of the taxine solution. The concentration-absorbance standard curve of taxine was then plotted. Figure 9 As shown, the concentration of free taxanein was calculated. The total weight was measured after cooling and drying.

[0086] (F) In vitro drug release: Drug release was determined by dialysis. A PBS solution (pH = 7.4) containing 0.5% Tween-80 was used as the release medium. Taxostatin was fully dissolved in ethanol to obtain a free taxostatin solution, which, along with the taxostatin liposome solution, was placed separately into dialysis bags (MWCO = 3500 Da), with a taxostatin concentration of 1 mg / mL. The dialysis bags were placed in beakers containing 50 mL of release medium, sealed, and placed on a constant-temperature shaker to observe drug release. The shaker operated at 37°C and 100 rpm. At time points of 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h, 600 μL of the release medium was taken and injected into 96-well plates for subsequent analysis, along with the same amount of fresh release medium. The absorbance of the release medium at each time point was detected using an ELISA reader. The concentration of taxanein in the release medium was calculated based on the plotted standard curve, and the cumulative release amount of taxanein was calculated. Figure 10The in vitro drug release curves are shown for an ethanol solution (Tax) containing taxine and magnetic liposomes loaded with taxine (Tax-BFe3O4-Lipo).

[0087] The results showed that in the first hour of release, the magnetic liposomes loaded with taxine released 18.47% of the taxine, while the ethanol solution containing taxine released 58.59% of the taxine. In the second hour of release, the magnetic liposomes loaded with taxine released 22.59% of the taxine, while the ethanol solution containing taxine released 78.41% of the taxine, approaching release equilibrium. At 72 hours, the magnetic liposomes loaded with taxine released 59.45% of the taxine, while the ethanol solution containing taxine released 80.58%, indicating that the liposomes had a sustained-release effect and could effectively release taxine.

[0088] (G) Cell Culture: Cortical neurons from neonatal Sprague-Dawley (SD) rats within 24 hours were dissected and initially cultured in vitro. Prefrontal cortex tissue was isolated and digested with 0.25% trypsin for 15–20 min, followed by the addition of 10% fetal bovine serum to stop digestion. After thorough pipetting, the mixture was centrifuged and resuspended in a cell suspension. Cells were then seeded in 6 cm culture dishes for subsequent experiments and placed in a 37°C incubator with 5% CO2. On day 5 of culture, an oxygen-glucose deprivation (OGD) model was established using a 5% CO2 anaerobic gas-generating bag (Anning bag) and an oxygen indicator to maintain a stable pH, creating a purely hypoxic extracellular environment. Under hypoxic and glucose-deficient conditions, mitochondrial function declined, oxygen free radicals increased, and an apoptosis cascade was initiated. The expression of apoptosis-related genes was also upregulated or downregulated accordingly. Figure 11 Bright-field images of normally cultured neurons. Figure 12 These are bright-field images of neurons 4 hours after OGD injury.

[0089] (H) Cytotoxicity Assay: Cell viability was assessed using the CCK-8 assay kit. The CCK-8 kit was diluted 1:9 to prepare the culture medium, and the mixture was incubated for 1 hour. Absorbance was measured at 450 nm using a fluorescence microplate reader (Thermo, USA) to determine cell viability. Groups were set up as a control group, an OGD group, a 20 μg / mL taxine liposome group (OGD+Tax-Lipo), and a 20 μg / mL magnetic liposome group loaded with taxine liposomes combined with a magnetic field (OGD+Tax-BFe3O4-Lipo) to explore the differences in the effects of the two liposome materials on cells. Figure 13As shown in the figure. The experimental results showed that, compared with the control group, the cell survival rate of the OGD group was significantly decreased (n=3, p<0.001). The OGD+Tax-Lipo group and the Tax-BFe3O4-Lipo combined magnetic field group were significantly improved compared with the OGD group (n=3, p<0.001), and there was a significant difference between the OGD+Tax-Lipo group and the Tax-BFe3O4-Lipo combined magnetic field group (n=3, p<0.05). This indicates that both the OGD+Tax-Lipo group and the Tax-BFe3O4-Lipo combined magnetic field group can significantly improve the survival rate of neurons, and the Tax-BFe3O4-Lipo combined magnetic field group is more effective than the OGD+Tax-Lipo group.

[0090] Each experiment was repeated at least three times, and all quantitative data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software, employing one-way ANOVA and t-tests. Statistical significance in the figures is denoted by *, **, and **, where * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0091] (J) Apoptosis Assay: Primary neurons were seeded on glass slides and subjected to oxygen-glucose deprivation treatment on day 5. After 4 hours of oxygen-glucose deprivation treatment, 20 μg / mL of taurine liposomes and 20 μg / mL of taurine-loaded magnetic liposomes were added to detect the neurorepair effect of the taurine-loaded magnetic liposomes. Samples were collected after 24 hours, and cells were washed three times with PBS for 5 minutes each time to remove impurities and cell debris. After washing, cells were fixed in 4% PFA (4% paraformaldehyde) at 4°C for 30 minutes. After fixation, cells were washed three times with PBS to remove excess PFA. Permeabilization was performed with 0.2% Triton X-100 and incubated at room temperature for 20 minutes. Cells were washed three times with PBS to remove excess 0.2% Triton X-100. A TdT reaction mixture was prepared in the dark, with a TdT reaction buffer:TdTenzyme ratio of 9:1. Add moistened cotton wool to a glass dish, invert the slide onto the TdT reaction solution, wrap it with aluminum foil, and incubate for 60 minutes. After incubation, wash the cells twice with PBS, each time for 5 minutes. Prepare a nuclear staining agent with a DAPI:PBS ratio of 1:2000 and incubate at room temperature for 5 minutes. After washing again with PBS, add anti-fluorescence quenching mounting solution to each slide, and acquire images using a laser confocal microscope. Figure 14 TUNEL staining results for cells treated with piperidine liposomes and magnetic liposomes loaded with piperidine. Figure 15This study presents the statistical results of cell apoptosis after treatment with piracetam liposomes and magnetic liposomes loaded with piracetam. The results showed that, compared to the control group, the OGD group had a significantly increased number of green-stained TUNEL-positive cells with stronger fluorescence intensity, all overlapping with the cell nucleus. The fluorescence intensity was reduced in the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined with magnetic field group, while the number of green-stained TUNEL-positive apoptotic cells was significantly decreased. Statistical results showed that the percentage of neuronal apoptosis in the OGD group was significantly higher than that in the control group (n=3, p<0.001). The percentage of neuronal apoptosis in the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined with magnetic field group was significantly lower than that in the OGD group. There was also a significant difference between the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined with magnetic field group (n=3, p<0.01). This indicates that the use of Tax-Lipo and Tax-BFe3O4-Lipo combined with magnetic field can significantly inhibit apoptosis, and the effect is better when Tax-BFe3O4-Lipo is used in combination with magnetic field, thus achieving the effect of nerve repair.

[0092] (K) PCR detection: RNA was extracted from the sample using an RNA extraction kit and reverse transcribed into cDNA. The gene expression of GAPDH, BAX, Caspase-3, and BCL-2 was then detected using a PCR instrument. Figure 16 , Figure 17 as well as Figure 18 The results show the Real-Time PCR detection of BAX, Caspase 3, and BCL2 in hypoxic nerve cells under the combined action of piracetam liposomes and magnetic liposomes loaded with piracetam, respectively, and a magnetic field. Figure 19 and Figure 20 These are the results of Real-Time PCR detection of NGF and BDNF in hypoxic neurons under the combined action of magnetic liposomes loaded with taurine and a magnetic field.

[0093] BAX and Caspase3 are pro-apoptotic genes that initiate the apoptosis program. Therefore, in this study, the expression of BAX and Caspase3 was significantly increased after 4 hours of hypoxic OGD treatment (n=3, p<0.05). Compared with the OGD+Tax-Lipo group, the OGD+Tax-BFe3O4-Lipo combined with magnetic field group showed a significant decrease in Bax expression in neurons of the OGD group (n=3, p<0.01). This indicates that both the Tax-Lipo group and the Tax-BFe3O4-Lipo combined with magnetic field can significantly inhibit Bax expression in neurons, and the OGD+Tax-BFe3O4-Lipo combined with magnetic field group had a more significant effect than the Tax-Lipo group (n=3, p<0.01). These results suggest that piperidin combined with magnetic particles and a magnetic field can more effectively inhibit the expression of the apoptosis gene BAX and promote neuronal growth.

[0094] Figure 17 The results showed that, compared with the control group, the expression level of Caspase3 was significantly increased after hypoxia injury (n=3, p<0.001), while the expression level of Caspase3 in the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined magnetic field group was significantly decreased compared with the OGD group (n=3, p<0.001). This indicates that both the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined magnetic field group can significantly inhibit the expression of Caspase3 in neurons, and the effect of the OGD+Tax-BFe3O4-Lipo combined magnetic field group is more significant (n=3, p<0.05). This suggests that the Tax-BFe3O4-Lipo combined magnetic field can significantly inhibit the expression of the apoptosis gene Caspase3 in neurons.

[0095] BCL2 is an anti-apoptotic gene that can prevent the initiation of the apoptosis program. Therefore, the worse the cell condition, the more pronounced the decrease in BCL2 expression, and vice versa. Figure 18 The results showed that the expression of BCL2 in the OGD group was significantly lower than that in the control group (n=3, p<0.001), while the expression in the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined with magnetic field group was significantly higher than that in the OGD group (n=3, p<0.001), and there was a significant difference between the two groups (n=3, p<0.001). This indicates that both the OGD+Tax-Lipo group and the Tax-BFe3O4-Lipo combined with magnetic field group can significantly promote the expression of the anti-apoptotic gene BCL2 in neurons, and the Tax-BFe3O4-Lipo combined with magnetic field group has a better effect.

[0096] In addition, to investigate the effects of piperidine liposomes and magnetic liposomes on neurogenesis in hypoxic neurons, real-time quantitative PCR was used to detect the gene expression of NGF and BDNF in cells. The results are as follows: Figure 19 and Figure 20 As shown:

[0097] Figure 19 As shown, the expression of NGF in the OGD damage group was significantly lower than that in the control group (n=3, P<0.05), and the expression in the OGD+Tax-BFe3O4-Lipo combined magnetic field group was significantly higher than that in the OGD damage group (n=3, P<0.01), and the increase in NGF expression was more significant than that in the OGD+Tax-Lipo group (n=3, P<0.05).

[0098] Figure 20 The results showed that, compared with the control group, the expression level of BDNF in neurons was significantly reduced under the action of OGD (n=3, P<0.05). Compared with the OGD group, the OGD+Tax-Lipo group and the OGD+Tax-BFe3O4-Lipo combined magnetic field group showed significantly increased BDNF expression, and the increase in BDNF expression in the OGD+Tax-BFe3O4-Lipo combined magnetic field group (n=3, P<0.01) was more significant than that in the OGD+Tax-Lipo group (n=3, P<0.05). This indicates that the combined use of Tax-Lipo and Tax-BFe3O4-Lipo not only inhibits apoptosis in hypoxic neurons but also significantly promotes growth, thus achieving a neuronal repair effect. The effect of the Tax-BFe3O4-Lipo combined magnetic field is more significant in comparison.

[0099] Example 2: Magnetic Tea Polyphenol Liposomes

[0100] Step 1: Fe3O4 magnetic nanoparticles were prepared by co-precipitation. 1.86 g of FeCl2·4H2O and 3.76 g of FeCl3·6H2O were magnetically stirred at 85 °C and 1000 rpm for 0.5 h. Then, 15 mL of 25% ammonium hydroxide was added. After stirring the mixture for 4 h, the product was collected, washed twice with deionized water, and twice with ethanol. Finally, it was dried in a freeze dryer at -50 °C for 22 h.

[0101] Step 2: To prevent particle aggregation and ensure stable dispersion of Fe3O4 magnetic nanoparticles, bovine serum albumin (BSA) was used to modify the magnetic nanoparticles. 60 mg of BSA was added to a solution containing 10 mg of Fe3O4 magnetic nanoparticles, and the solution was vigorously stirred at 0°C for 10 hours to obtain a BFe3O4 solution.

[0102] Step 3: Dissolve 60mg egg yolk lecithin, 20g cholesterol, 20mg distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 6mg tea polyphenols in 50mL of ethanol solution. Vortex mix for 2min, then transfer the mixture to a 100mL round-bottom flask in a rotary evaporator. Remove the ethanol by rotary evaporation under reduced pressure at 50℃ and 100rpm, ultimately forming a uniform lipid film at the bottom of the rotary evaporator flask.

[0103] Step 4: Add 6 mL of BFe3O4 solution to the rotary evaporator for hydration, and sonicate until the lipid film at the bottom of the evaporator is completely detached. After rotating the suspension, incubate it in a 40°C water bath with continuous stirring. Use a pipette to blow the suspension through the evaporator and collect the lipid suspension. Transfer the collected suspension to a 10 mL sample vial and sonicate it for 5 min using a cell disruptor probe at 200 W on ice. The sonication time is 3 s, followed by a 2 s pause. Finally, filter the product to obtain a clear and homogeneous magnetic tea polyphenol-loaded liposome solution.

[0104] Example 3: Magnetic Resveratrol Liposomes

[0105] Step 1: Fe3O4 magnetic nanoparticles were prepared by co-precipitation. 1.12 g of FeCl2·4H2O and 2.99 g of FeCl3·6H2O were magnetically stirred at 85 °C and 600 rpm for 0.5 h. Then, 10 mL of 25% ammonium hydroxide was added. After stirring the mixture for 3.5 h, the product was collected, washed twice with deionized water, and twice with ethanol. Finally, it was dried in a freeze dryer at -50 °C for 20 h.

[0106] Step 2: To prevent particle aggregation and ensure stable dispersion of Fe3O4 magnetic nanoparticles, bovine serum albumin (BSA) was used to modify the magnetic nanoparticles. 40 mg of BSA was added to a solution of 10 mg of Fe3O4 magnetic nanoparticles, and the mixture was vigorously stirred at 0°C for 10 hours to obtain a BFe3O4 solution.

[0107] Step 3: Dissolve 50 mg of egg yolk lecithin, 15 mg of cholesterol, 15 mg of DSPE-PEG2000, and 8 mg of resveratrol in 10 mL of ethanol solution. Vortex mix for 2 min, then transfer the mixture to a 100 mL round-bottom flask in a rotary evaporator. Remove the ethanol by rotary evaporation under reduced pressure at 50 °C and 100 rpm, ultimately forming a uniform lipid film at the bottom of the rotary evaporator flask.

[0108] Step 4: Add 5 mL of BFe3O4 solution to the rotary evaporator for hydration, and sonicate until the lipid film at the bottom of the evaporator is completely detached. Then, incubate the evaporator in a 40°C water bath with continuous stirring, and use a pipette to blow the mixture into the evaporator to collect the lipid suspension. Transfer the collected suspension to a 10 mL sample vial, and sonicate it for 3 minutes at 250 W using a cell disruptor probe under ice conditions. Sonicate for 3 seconds, pause for 2 seconds, and finally filter the product to obtain a clear and homogeneous magnetic resveratrol-loaded liposome solution.

Claims

1. A method for preparing magnetic drug-loaded liposomes for nerve repair, characterized in that, Includes the following steps: The target components of the design are: egg yolk lecithin, cholesterol, and DSPE-MPEG2000 in 1 mL of liposome solution. The mass fractions of the drugs were 0.5%–1.5%, 0.15%–0.35%, 0.15%–0.35%, 0.1%–0.2%, and 0.1%–0.2%, respectively. Step 1: Preparation using the co-precipitation method Magnetic nanoparticles; Add to deionized water and After magnetic stirring at a water bath temperature of 70℃~85℃ for 0.5h, ammonium hydroxide was added, and stirring was continued for 3h~4h. The product was then removed, washed twice with deionized water, and twice with ethanol. After washing, the product was dried in a freeze dryer at -30℃~-50℃ for 18h~24h to obtain the product. Magnetic nanoparticles; Dosage: 1g to 3g is needed per 100mL of deionized water. 2g to 4g 10 mL to 20 mL of ammonium hydroxide; The ammonium hydroxide has a mass percentage concentration of 25%. The magnetic stirrer's speed is adjustable from 400 rpm to 1000 rpm; Step 2: Modification with magnetic nanoparticles; Add bovine serum albumin to The magnetic nanoparticle solution was stirred vigorously at 0°C for 10 hours to obtain... Solution; Dosage: 10mg Add 30mg to 80mg of bovine serum albumin to magnetic nanoparticles; The magnetic stirrer's speed is adjustable from 800 rpm to 1200 rpm; Step 3: Prepare lipid films by rotary evaporation under reduced pressure; Egg yolk lecithin, cholesterol, DSPE-PEG2000 and drugs were dissolved in an ethanol solution and ultrasonically vortexed for 2 minutes. The mixture was then transferred to a round-bottom flask of a rotary evaporator and the ethanol was removed by rotary evaporation under reduced pressure at 50°C and 50-100 rpm. Finally, a uniform lipid film was formed at the bottom of the round-bottom flask. The drug is a natural antioxidant used for nerve repair; specifically, the drug is one or more of piperidine or tea polyphenols. Dosage: 30mg-90mg of egg yolk lecithin, 5mg-15mg of cholesterol, 5mg-15mg of DSPE-PEG2000, and 3mg-9mg of the drug are required in 50mL of ethanol; The ultrasonic frequency is 60kHz; DSPE-PEG2000 refers to distearylphosphatidylethanolamine-polyethylene glycol 2000; Step 4: Liposomes are obtained after hydration; Add to the rotary evaporating flask The solution was hydrated and sonicated until the lipid film at the bottom of the rotary evaporator was completely detached. Then, it was placed in a water bath at 30℃~60℃ and stirred continuously. The rotary evaporator was blown with a pipette to collect the lipid suspension. The collected suspension was transferred to a sample bottle and sonicated at 0℃ using the probe of a cell disruptor for 3min~10min, i.e., sonication for 3s, stop for 2s. Finally, the product was filtered to obtain a clear and homogeneous magnetic drug-loaded liposome solution. The ultrasonic frequency is 60kHz; The magnetic stirrer's speed is adjustable from 350 rpm to 600 rpm; The power of the cell disruptor is 150W to 300W; The prepared magnetic drug-loaded liposomes have a phospholipid bilayer structure, are surface-modified with PEG, and encapsulate magnetic nanoparticles and drugs.

Images