Asparagine endopeptidase inhibitor neuronal targeting liposome and preparation method thereof

Abstract

The invention relates to an asparagine endopeptidase inhibitor neuronal targeting liposome and a preparation method thereof. The liposome comprises the following components: lecithin, cholesterol, phospholipid polyethylene glycol polypeptide, a fluorescent dye and an asparagine endopeptidase inhibitor. The preparation method has relatively good drug loading capacity and entrapment efficiency; after being atomized and dosed through a nose, the nebulizer can smoothly enter the brain of a mouse through a blood brain barrier to be distributed, and has good biocompatibility; according to the invention, DNA damage and ROS reaction of irradiated brain neurons and glial cells can be reduced, and the research and development value and important academic significance of the liposome for assisting in improving radiation brain injury are revealed.

Description

Technical Field

[0001] The invention belongs to the field of new medical materials, and particularly relates to an asparagine endopeptidase inhibitor neuron-targeted liposome and a preparation method thereof. Background Art

[0002] Liposome technology is an important breakthrough in the field of drug delivery in recent years, especially in improving the bioavailability of drugs, reducing side effects, and achieving targeted therapy. Liposomes are nanoscale spherical structures composed of natural or synthetic lipids that can encapsulate drug molecules. Through their unique lipid bilayer structure, they effectively protect drugs from rapid decomposition by enzymes in the body and prolong their circulation time in the body. Liposomes are widely used in many medical fields, including cancer treatment, anti-infection treatment, and vaccine delivery. Especially in cancer treatment, liposomes can effectively deliver chemotherapy drugs directly to tumor cells, significantly enhancing the therapeutic effect while reducing the toxic effects on normal cells. Liposomes can provide a relatively stable environment for drug molecules to prevent rapid degradation in the blood circulation. By encapsulating drugs, liposomes can reduce the possibility of drugs being dispersed into non-targeted areas in the body and reduce side effects. Liposomes can achieve targeted drug delivery through specific formulation design (such as surface modification), increase the concentration of drugs at the lesion, and enhance the therapeutic effect. Liposomes can be delivered through different administration methods, including intravenous injection, intramuscular injection, etc., providing more clinical application options.

[0003] Radiotherapy is an important adjuvant treatment for patients with head and neck tumors such as nasopharyngeal carcinoma, primary or metastatic brain tumors, however, radiotherapy can also cause damage to adjacent normal brain tissue, leading to central nervous system complications. Radiation-induced brain injury (RIBI) is the most serious complication of radiotherapy for patients with head and neck tumors. Its clinical symptoms include brain edema, necrosis, and cognitive dysfunction, which seriously affect the quality of life of patients. Currently, many literatures have revealed that asparagine endopeptidase can participate in the injury process after radiation in various ways. However, due to the lack of understanding of the pathogenesis of RIBI and the lack of active and effective therapeutic drugs, the clinical treatment options for RIBI patients are relatively limited and further research is needed. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an asparagine endopeptidase inhibitor neuron-targeted liposome and a preparation method thereof, wherein the liposome can reduce DNA damage and ROS response of brain neurons and glial cells after irradiation, and reveal that the liposome can have research and development value and important academic significance in assisting in improving radiation-induced brain damage.

[0005] The present invention provides an asparagine endopeptidase inhibitor neuron-targeted liposome, which comprises the following components: lecithin, cholesterol, phospholipid polyethylene glycol polypeptide, fluorescent dye and asparagine endopeptidase inhibitor; wherein the phospholipid polyethylene glycol polypeptide is used to increase the solubility and stability of the asparagine endopeptidase inhibitor and play a role in reducing immunogenicity.

[0006] Preferably, the lecithin is soybean lecithin with a molecular weight of 20-100 kDa.

[0007] Preferably, the molecular weight of cholesterol is 25-60 kDa.

[0008] Preferably, the fluorescent dye is CY5.

[0009] Preferably, the mass ratio of the lecithin, cholesterol, phospholipid polyethylene glycol polypeptide, fluorescent dye and asparagine endopeptidase inhibitor is 200-300:50-100:5-10:1-2:4-5.

[0010] Furthermore, the liposome is administered nasally via an atomizer. Furthermore, the liposome is formulated into a suspension for administration.

[0011] The present invention also provides a method for preparing neuron-targeted liposomes of asparagine endopeptidase inhibitor, comprising the following steps:

[0012] (1) dissolving lecithin, cholesterol, phospholipid polyethylene glycol polypeptide and fluorescent dye in chloroform, evaporating under reduced pressure to form a film, and adding an aqueous solution of asparagine endopeptidase inhibitor to hydrate;

[0013] (2) The obtained product is treated with ultrasound and a liposome extruder; dialyzed, supplemented with deionized water to a fixed volume, and evenly dispersed to obtain asparagine endopeptidase inhibitor neuron-targeted lipids.

[0014] The present invention also provides an application of asparagine endopeptidase inhibitor neuron-targeted liposomes in the preparation of radiation brain injury drugs.

[0015] Beneficial Effects

[0016] The invention has good drug loading capacity and encapsulation rate; after atomized nasal administration, it can smoothly pass through the blood-brain barrier and enter the mouse brain for distribution, and has good biocompatibility; the invention can reduce DNA damage and ROS response of brain neurons and glial cells after irradiation, revealing that the liposome can have research and development value and important academic significance in assisting in improving radiation brain damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is a particle size detection diagram and a Zeta potential detection diagram of the liposome of the present invention.

[0018] Figure 1 B is the cumulative release curve of the liposome of the present invention in an in vitro environment.

[0019] Figure 2 This is a diagram showing the distribution of drugs in mouse brain slices after liposome aerosol administration of the present invention.

[0020] Figure 3 This is the HE staining picture of the drug in mice after the liposome aerosol administration of the present invention.

[0021] Figure 4 γH2AX immunofluorescence was used to observe the effects of different treatments on double-strand DNA damage in microglia.

[0022] Figure 5 Fluorescence images of ROS generated in neural stem cells of different treatment groups. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0024] Example 1

[0025] Preparation of neuron-targeted liposomes containing asparagine endopeptidase inhibitors.

[0026] 1. Preparation of lipid mixture and lipid film

[0027] Ingredients and Dissolution:

[0028] Soy lecithin: about 200 mg;

[0029] Cholesterol: about 50 mg (the ratio of lecithin to cholesterol is about 4:1);

[0030] DSPE-PEG2000-RVG29: about 5 mg;

[0031] CY5: about 1 mg;

[0032] After weighing the above components, 5 mL of chloroform was added to completely dissolve them.

[0033] Formation of lipid membrane:

[0034] Evaporation was performed using a rotary evaporator at 45 °C to ensure complete removal of chloroform.

[0035] The rotation speed is controlled at 100-120 rpm for about 10-15 minutes until a uniform lipid film is formed.

[0036] Hydration treatment:

[0037] The concentration of the aqueous solution of AEP inhibitor (purchased from MCE, catalog number: HY-114174) used for hydration was about 2 mg / mL, and the total volume was 2 mL.

[0038] The hydration temperature was controlled at 60°C and the hydration time was about 2 hours to ensure that the lipid membrane was fully hydrated to form liposomes.

[0039] 2. Ultrasonic and extrusion treatment

[0040] Sonication:

[0041] The ultrasound frequency was set to 20 kHz and the power to 100 W.

[0042] The ultrasonic treatment time was about 5 min, and a cold water bath was used to prevent heating of the sample.

[0043] Liposome extrusion:

[0044] During the extrusion process, the liposomes were passed through a polycarbonate membrane with a pore size of 200 nm and the extrusion was repeated 10 times to ensure the consistency of particle size.

[0045] The temperature was maintained at room temperature during the extrusion process.

[0046] 3. Dialysis to remove unloaded drugs

[0047] Dialysis:

[0048] Dialysis was performed using a polycarbonate membrane (pore size 30 nm).

[0049] During the dialysis process, the dialysate is a large amount of deionized water, which is replaced every 2 hours and the dialysis lasts for 24 hours.

[0050] 4. Volume determination and final processing

[0051] Add deionized water to make up to volume:

[0052] Adjust the volume to the nearest 10 mL based on the volume after dialysis.

[0053] 5. Mixing and Dispersion

[0054] Final mixing was performed using a high shear mixer set at 5000 rpm for 10 minutes.

[0055] Example 2

[0056] Particle size, Zeta potential detection, in vitro release and encapsulation efficiency detection of asparagine endopeptidase inhibitor neuron-targeted liposomes.

[0057] The particle size and Zeta potential of the liposomes were measured using a nanoparticle size potentiometer (NanoBrook 90plus PALS, Brookhaven, USA) at 25°C and a light scattering angle of 90°. 20 mL of the asparagine endopeptidase inhibitor neuron-targeted liposomes prepared in Example 1 were taken with a pipette and diluted 100 times with ultrapure water for particle size and Zeta potential detection.

[0058] Figure 1 a shows that the average particle size of the asparagine endopeptidase inhibitor neuron-targeted liposomes prepared in Example 1 of the present invention is 120.5±1.86 nm, and the potential is -21.88±1.52 mV.

[0059] The release of asparagine endopeptidase inhibitor neuron-targeted liposomes was detected by dialysis. Asparagine endopeptidase inhibitor neuron-targeted liposomes and an equal concentration of asparagine endopeptidase inhibitor solution were placed in a dialysis bag (molecular weight cutoff Mn=3kDa) soaked in PBS and the two ends were tied tightly, and then placed in 30ml of PBS (phosphate buffered saline) dialysate containing 0.5% Tween-80. Samples were taken at fixed time points (2, 4, 6, 8, 12, 24, 48, 72h) and an equal amount of fresh dialysate was added in time. The samples were analyzed for the content of asparagine endopeptidase inhibitor by high performance liquid chromatography. Figure 1 b The data show that drug release was relatively active in the first 30 hours, and then stabilized until the end of the 48-hour observation period, when the release amount approached a steady state.

[0060] Example 3

[0061] The encapsulation efficiency of neuron-targeted liposomes containing asparagine endopeptidase inhibitor was determined by high performance liquid chromatography.

[0062] The first step is to draw a standard curve of asparagine endopeptidase inhibitors. The chromatographic conditions are as follows: a Phenomenex Gemini 5μm C18110A chromatographic column is used, the UV detection wavelength is set to 230nm, the column temperature is 37°C, and the mobile phase is 75:25 (v / v) methanol and water. Under this condition, a series of concentration gradient asparagine endopeptidase inhibitor standards (concentrations of 0.02, 0.04, 0.06, 0.08, 0.09, and 0.1μg / μL) prepared with chromatographic methanol are injected in sequence, and each concentration is injected 3 times. The chromatographic peaks are recorded and integrated and analyzed. The peak area (Y) and mass concentration (X) are used to make a standard curve. Linear regression is performed to obtain the standard curve equation: y=7185.4x-11819, R2=0.9999.

[0063] The second step is to detect the encapsulation rate of asparagine endopeptidase inhibitor in neuron-targeted liposomes of asparagine endopeptidase inhibitor. Take 500 μL of neuron-targeted liposomes of asparagine endopeptidase inhibitor, add 2 mL of methanol to fully dissolve, filter, inject under the same chromatographic conditions as the above standard curve, record the chromatogram, integrate and analyze it, and calculate by the standard curve equation to obtain the total amount of drug in the liposome; take 100 μL of liquid centrifuged in an ultrafiltration centrifuge tube, add 400 μL of methanol, filter and inject according to the above conditions, record the chromatogram, integrate and analyze it, and calculate by the standard curve equation to obtain the free asparagine endopeptidase inhibitor in the liposome. The encapsulation rate is calculated as follows: encapsulation rate = (total amount of drug - free amount of drug) / total amount of drug × 100%.

[0064] The encapsulation efficiency of the asparagine endopeptidase inhibitor neuron-targeted liposomes prepared in Example 1 was 78%.

[0065] Example 4

[0066] The aerosol administration of neuron-targeted liposome suspension of asparagine endopeptidase inhibitor was tested, and the distribution and biocompatibility of the drug in the mouse brain were tested.

[0067] In this part, 10 C57B6 / L male mice aged 6-8 weeks were intraperitoneally injected with 0.9% pentobarbital anesthetic. After the plantar reflex disappeared, the neuron-targeted liposome suspension of asparagine endopeptidase inhibitor was aerosolized and then irradiated with 40Gy of whole brain. After 1 hour, the mice were killed by cervical dislocation and brain tissue was obtained for preparation of frozen sections. The specific implementation steps are as follows:

[0068] 1. Fixation. Take the brain by perfusion and fix it by immersion in paraformaldehyde;

[0069] 2. Dehydration. Use 15%, 20%, and 30% PB sucrose solution to dehydrate the brain tissue in a gradient manner;

[0070] 3. Frozen sections. Take out the brain tissue and embed it with OCT glue, layer by layer. Note: There should be no bubbles or gaps, otherwise the brain tissue will be frozen. The temperature of frozen sections can be between -18℃ and -25℃, and the temperature of brain tissue is best at -18℃.

[0071] 4. Frozen sections of 8-10 μm should be placed at room temperature for more than 30 minutes to prevent peeling and stored at -20°C. Frozen sections taken out of the refrigerator should be placed at room temperature for more than 30 minutes before immunofluorescence.

[0072] 5. Wash three times with 1xPBS, 3-5 minutes each time.

[0073] 6. Block with 2% BSA at room temperature or 37°C for 1 hour.

[0074] 7. Incubate with primary antibody at 4°C overnight. Wash 3 times with 1xPBS, 3-5 minutes each time.

[0075] 8. Incubate with secondary antibody at room temperature or 37°C in the dark for 1 hour. Wash 3 times with 1xPBS, 3-5 minutes each time.

[0076] 9. DAPI staining: Wash 3 times with 1xPBS, 3-5 minutes each time.

[0077] 10. Add anti-quenching agent and seal the slide.

[0078] Figure 2 The results showed that the neuron-targeted liposome suspension of asparagine endopeptidase inhibitor could successfully pass through the blood-brain barrier and enter the brain after aerosol administration.

[0079] Example 5

[0080] In this part, 10 C57B6 / L male mice aged 6-8 weeks were injected with 0.9% pentobarbital anesthetic via intraperitoneal injection. After the plantar reflex disappeared, the neuron-targeted liposome suspension of asparagine endopeptidase inhibitor was aerosolized and administered once a day for 14 consecutive days. After 14 days, the mice were killed and the main organs were stained with hematoxylin and eosin (H&E). The specific implementation steps are as follows:

[0081] 1. Dewax the paraffin sections to water: wash the sections in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, and distilled water in sequence.

[0082] 2. Hematoxylin staining of cell nuclei: Stain the sections with hematoxylin solution for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia solution, and rinse with running water.

[0083] 3. Eosin staining of cytoplasm: Stain the sections in eosin staining solution for 1-3 minutes.

[0084] 4. Dehydration and sealing: Place the slices in 95% alcohol I, 95% alcohol II, anhydrous ethanol I, anhydrous ethanol II, xylene I, and xylene II in turn to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0085] 5. Microscope examination, image acquisition and analysis.

[0086] Figure 3 Histological analysis showed that no tissue or cell damage was observed in all organs after administration of the neuron-targeted liposome suspension of asparagine endopeptidase inhibitor.

[0087] Example 6

[0088] The effect of asparagine endopeptidase inhibitor neuron-targeted liposome suspension on DNA damage of microglia under irradiation was examined.

[0089] The specific implementation steps are as follows:

[0090] The cells were cultured to the logarithmic growth phase, and equal amounts of cells were irradiated and then plated on slides. The cells were incubated on ice in PBS containing 5% BSA for 20 minutes and then blocked with 5% donor bovine serum for 20 minutes. After blocking, the cells were treated with primary antibody (γH2AX antibody), and then treated with fluorescence-coupled secondary antibody. The γH2AX fluorescence signal was captured and quantified using a confocal microscope. The number of γH2AX Foci in 100 cell nuclei was counted in each group. Those greater than 5 were considered positive, and the positive cells were statistically analyzed.

[0091] Figure 4 The results showed that under irradiation, the neuron-targeted liposome suspension group of asparagine endopeptidase inhibitor could significantly reduce DNA double-strand damage compared with the control group.

[0092] Example 7

[0093] To examine the effect of asparagine endopeptidase inhibitor neuron-targeted liposome suspension on ROS damage of neural stem cells under irradiation.

[0094] The specific implementation steps are as follows:

[0095] The cells were washed three times with PBS, and then fixed with 4% paraformaldehyde fixative for 10 min; washed three times with PBS, and treated with 0.1%-0.3% Triton-X100 for 15 min; washed three times with PBS, blocked with blocking solution (10% goat serum or 1%-3% bovine serum albumin) for 1 h, and incubated with primary antibody at 1:100 overnight at 4°C; washed three times with PBS, added with secondary antibody, and incubated at room temperature for 1 h; washed three times with PBS, and incubated with Hoechst 33342 or PI at room temperature for 5 min; washed with PBS, and sealed, and then observed and photographed with a laser confocal microscope (Leica SP2) or a fluorescence microscope.

[0096] Figure 5 The results showed that under irradiation, the neuron-targeted liposome suspension group of asparagine endopeptidase inhibitor could significantly reduce ROS damage compared with the control group.

Claims

1. An asparagine endopeptidase inhibitor neuron-targeted liposome, characterized in that: The liposome comprises the following components: phosphatidylcholine, cholesterol, phospholipid polyethylene glycol polypeptide, fluorescent dye and asparagine endopeptidase inhibitor.

2. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 1, characterized in that: The lecithin is soybean lecithin with a molecular weight of 20-100 kDa.

3. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 1, characterized in that: The molecular weight of cholesterol is 25-60 kDa.

4. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 1, characterized in that: The fluorescent dye is CY5.

5. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 1, characterized in that: The mass ratio of the lecithin, cholesterol, phospholipid polyethylene glycol polypeptide, fluorescent dye and asparagine endopeptidase inhibitor is 200-300:50-100:5-10:1-2:4-5.

6. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 1, characterized in that: The liposomes are administered nasally via a nebulizer.

7. The neuron-targeted liposome of asparagine endopeptidase inhibitor according to claim 6, characterized in that: The liposomes are formulated for administration as a suspension.

8. A method for preparing neuron-targeted liposomes of asparagine endopeptidase inhibitor, comprising the following steps: (1) dissolving lecithin, cholesterol, phospholipid polyethylene glycol polypeptide and fluorescent dye in chloroform, evaporating under reduced pressure to form a film, and adding an aqueous solution of asparagine endopeptidase inhibitor to hydrate; (2) The obtained product is treated with ultrasound and a liposome extruder; dialyzed, supplemented with deionized water to a fixed volume, and evenly dispersed to obtain asparagine endopeptidase inhibitor neuron-targeted liposomes.

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