Brain-targeted active oxygen responsive hydrogen sulfide donor liposome as well as preparation method and application thereof

Abstract

The invention discloses a brain-targeted active oxygen responsive hydrogen sulfide donor liposome, a preparation method and an application, and belongs to the technical field of pharmaceutical preparations, the structure of the brain-targeted active oxygen responsive hydrogen sulfide donor liposome comprises a lipid material and a hydrogen sulfide donor encapsulated in the lipid material, the lipid material comprises phospholipid, cholesterol, active oxygen sensitive lipid and brain-targeted lipid, and the lipid material comprises phospholipid, cholesterol, active oxygen sensitive lipid and brain-targeted lipid. The active oxygen sensitive lipid comprises a phospholipid lipophilic part, a polyethylene glycol hydrophilic part and an active oxygen sensitive bond, and the brain-targeted lipid comprises a phospholipid lipophilic part, a polyethylene glycol hydrophilic part and brain-targeted peptide. The hydrogen sulfide donor lipidosome can penetrate through a blood brain barrier, target neuronal cells, respond to an active oxygen environment of a focus part and sensitively release a hydrogen sulfide donor, so that hydrogen sulfide is distributed in the focus neuronal cells, active oxygen and active nitrogen substances are effectively removed, oxidation and nitration stress are inhibited, and damage and death of the neuronal cells are reduced; the growth and functional recovery of neuronal cells are promoted, and the application prospect in the aspect of treating central nervous system injury diseases is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome, a preparation method and an application thereof. Background Art

[0002] Hydrogen sulfide is an endogenous gaseous neurotransmitter and has important physiological effects on the nervous system. Hydrogen sulfide is usually produced by cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase during the metabolism of sulfur-containing amino acids in the central nervous system. Hydrogen sulfide mainly participates in three reactions in organisms: binding to the metal center of proteins and regulating redox reactions, scavenging reactive oxygen and reactive nitrogen species, and mediating S-sulfhydration modification of proteins (conversion of the R-SH group of proteins into the R-SSH group). In the nervous system, hydrogen sulfide is metabolized to persulfide, sulfite, thiosulfate, sulfate and polysulfide through an oxidative pathway. In addition to oxidative metabolism, hydrogen sulfide forms persulfide through S-sulfhydration modification of proteins and is stored in proteins in this form. A decrease in the content of endogenous hydrogen sulfide will lead to a reduction in the S-sulfhydration modification of proteins, affecting their normal catalytic activity or functional characteristics, accumulation of reactive oxygen and reactive nitrogen species, exacerbating mitochondrial function damage, generating cell inflammation, and causing cell damage, etc. Literature reports also show an imbalance in the content of hydrogen sulfide in most central nervous system injury diseases. At the same time, central nervous system injury diseases are closely related to neuroinflammation, and oxidative and nitrative stress release excessive reactive oxygen and reactive nitrogen species, further increasing neuronal cell damage and neuroinflammation.

[0003] Parkinson's disease is a neurological disease with a significant upward trend in prevalence, and the clinical treatment is mainly dopamine replacement therapy. Dopamine replacement therapy can only improve the motor symptoms of patients, cannot relieve, prevent or reverse the disease progression, and has a short drug half-life and patients are prone to behavioral fluctuations. As the disease progresses, most patients supplemented with dopamine often develop severe movement disorder complications within 3-5 years.

[0004] The S-sulfhydration modification of protein cysteine residues by hydrogen sulfide plays an important role in the pathophysiology of Parkinson's disease. Literature reports show that the S-sulfhydration modification of the ubiquitin ligase Parkin in the striatum of Parkinson's disease patients is significantly decreased, resulting in the dysfunction of the ubiquitin-proteasome system in clearing abnormal α-synuclein aggregates, and the accumulated abnormal α-synuclein aggregates further exacerbate oxidative and nitrative stress, damaging neuronal cells. Therefore, it is necessary to supplement hydrogen sulfide gas to damaged neuronal cells to restore their hydrogen sulfide level to the normal physiological state, and effectively treat Parkinson's disease by inhibiting oxidative and nitrative stress and inhibiting neuroinflammation.

[0005] Exogenous hydrogen sulfide donors have currently been used in the treatment of Parkinson's disease, including hydrogen sulfide gas, sodium hydrosulfide, and allicin, etc. However, there are still some challenges in the fields of its treatment research and application. For example, there are severe technical and safety challenges with hydrogen sulfide gas, and the concentration and inhalation dose of hydrogen sulfide gas need to be precisely monitored in real time; water-soluble hydrogen sulfide donors (such as sodium hydrosulfide) have a fast release rate, there is a sudden release effect of hydrogen sulfide in the body, and the half-life is less than ten minutes; sustained-release hydrogen sulfide donors (such as GYY4137) have defects in targeted delivery to the central nervous system, lack the ability to distribute in the lesion area of Parkinson's disease, and have a low efficiency of entering the brain; thiol-activated hydrogen sulfide donors (such as organosulfur compounds derived from garlic, including allicin, diallyl disulfide, diallyl trisulfide, and S-allylcysteine) are restricted by the thiol level in the body. The research on the hydrogen sulfide donor delivery system is a frontier direction worthy of exploration at the present stage.

[0006] The Chinese patent document with the publication number CN104109145A discloses a class of hydrogen sulfide donor derivatives based on flavonoid structures and their applications in the treatment of neuroinflammation-related diseases. The hydrogen sulfide donor compounds based on flavonoid structures provided by this invention exert the synergistic effect of hydrogen sulfide and flavonoid derivatives, improve the anti-neuroinflammatory effect of the compounds, and thus can treat central nervous system diseases related to neuroinflammation, such as the treatment of Alzheimer's disease and Parkinson's disease. The Chinese patent document with the publication number CN102078327A discloses the uses of hydrogen sulfide donor sodium hydrosulfide, allylcysteine, and their analogs in the preparation of drugs for treating central nervous system diseases. However, the hydrogen sulfide donors applied above are non-specifically released in the blood circulation, and the efficiency of entering the brain is limited; there are defects in the hydrogen sulfide donors. For example, sodium hydrosulfide releases hydrogen sulfide too fast, and the release of hydrogen sulfide by allylcysteine is restricted by the thiol level in the body. Therefore, it is necessary to select appropriate exogenous hydrogen sulfide donors and design a drug delivery system that targets the brain, responds to the reactive oxygen species environment at the lesion site, and sensitively releases hydrogen sulfide donors, so that hydrogen sulfide can be distributed in the lesion neuron cells. Summary of the Invention

[0007] The present invention provides a brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome. This liposome uses phospholipids, cholesterol, brain-targeting lipids, and reactive oxygen species-sensitive lipids to encapsulate hydrogen sulfide donors, and can cross the blood-brain barrier, target lesion neuron cells, respond to the reactive oxygen species environment at the lesion site, and sensitively release hydrogen sulfide donors. Compared with free hydrogen sulfide donors, this liposome can achieve efficient brain-targeted delivery, improve the distribution of hydrogen sulfide donors in lesion neuron cells, and improve neuroinflammation by inhibiting oxidative and nitrative stress, effectively reversing the death of dopaminergic neurons in the nigrostriatal pathway.

[0008] The specific technical solutions adopted are as follows:

[0009] A brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome, the structure of which includes a lipid material and a hydrogen sulfide donor encapsulated within the lipid material. The lipid material includes phospholipids, cholesterol, reactive oxygen species-sensitive lipids, and brain-targeting lipids. The reactive oxygen species-sensitive lipids include a phospholipid lipophilic moiety, a polyethylene glycol hydrophilic moiety, and a reactive oxygen species-sensitive bond. The brain-targeting lipids include a phospholipid lipophilic moiety, a polyethylene glycol hydrophilic moiety, and a brain-targeting peptide.

[0010] Preferably, the hydrogen sulfide donor is GYY4137 (abbreviated as GYY). GYY is a water-soluble slow-release organic donor of hydrogen sulfide. GYY has pH-dependence and temperature-dependence. Under the conditions of 37 °C, pH 7.4 PBS, it begins to hydrolyze and release hydrogen sulfide within 15 minutes and reaches equilibrium at 75 minutes.

[0011] Preferably, the phospholipids are selected from distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, or phosphatidylcholine; the reactive oxygen species-sensitive bond in the reactive oxygen species-sensitive lipids is a ketothiol bond; the brain-targeting peptide in the brain-targeting lipids is the RVG29 peptide.

[0012] The ketothiol bond is a reactive oxygen species-sensitive bond that can react with reactive oxygen species, resulting in the cleavage of the chemical bond and the destruction of the liposome structure, thereby achieving drug release.

[0013] The RVG29 peptide is a brain-targeting peptide derived from the rabies virus glycoprotein that specifically binds to the nicotinic acetylcholine receptor. Through receptor-mediated endocytosis, RVG29 effectively crosses the blood-brain barrier and targets neurons.

[0014] More preferably, the reactive oxygen species-sensitive lipids are selected as distearoyl phosphatidylethanolamine-ketothiol-polyethylene glycol (which can be directly purchased), and the brain-targeting lipids are selected as distearoyl phosphatidylethanolamine-polyethylene glycol-RVG29 (synthesized according to the records of the prior art).

[0015] Preferably, the particle size of the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome is ≤300 nm, the drug loading is 3 wt% - 6 wt%, and the encapsulation efficiency is 45 wt% - 65 wt%.

[0016] The present invention also provides a preparation method of the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome. Using the thin film dispersion method, it specifically includes the following steps:

[0017] S01 Dissolve the hydrogen sulfide donor and the lipid material in an organic solvent to obtain an organic phase;

[0018] S02 Rotavaporize the organic phase under heating conditions to obtain an organic phase thin film. Add water to the organic phase thin film, hydrate, ultrasonicate, and ultrafiltrate and centrifuge to obtain the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome.

[0019] The thin film dispersion method has a better encapsulation effect than the reverse evaporation method, the double emulsion method, etc.

[0020] Preferably, the organic solvent is a mixed solution of dichloromethane and methanol.

[0021] Preferably, in step S01, the feeding mass ratio of the hydrogen sulfide donor to the lipid material is 0.5-2:11; in the lipid material, the feeding mass ratio of phospholipid, cholesterol, reactive oxygen-sensitive lipid and brain-targeting lipid is 6-8:1-3:1:1.

[0022] More preferably, the feeding mass ratio of the hydrogen sulfide donor to the lipid material is 1:11; in the lipid material, the feeding mass ratio of phospholipid, cholesterol, reactive oxygen-sensitive lipid and brain-targeting lipid is 7:2:1:1. Under the conditions of the above feeding ratio, it is more helpful for the preparation of the hydrogen sulfide donor liposome.

[0023] The present invention also provides a therapeutic drug for central nervous system injury diseases, including the brain-targeting reactive oxygen-responsive hydrogen sulfide donor liposome described above.

[0024] Specifically, the central nervous system injury diseases include but are not limited to Parkinson's disease.

[0025] Preferably, the administration method of the therapeutic drug for central nervous system injury diseases is intravenous injection, and the dosage is 65-130 μM / kg (mouse), preferably, the dosage is 130 μM / kg.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The brain-targeting reactive oxygen-responsive hydrogen sulfide donor liposome provided by the present invention, the RVG29 peptide in the brain-targeting lipid specifically targets the nicotinic acetylcholine receptor, improves the brain entry efficiency of the hydrogen sulfide donor, and realizes the brain-targeting function. The ketothiol bond in the reactive oxygen-sensitive lipid regulates the reactive oxygen environment-sensitive release of the hydrogen sulfide donor in the diseased neurons, increases the hydrogen sulfide concentration at the lesion site, and realizes the reactive oxygen-responsive drug release function. The hydrogen sulfide donor is encapsulated by liposomes, and through the synergistic effect of each component, the transmembrane transport efficiency of the hydrogen sulfide donor across the blood-brain barrier is improved, the hydrogen sulfide donor is released in response to the reactive oxygen environment at the lesion site, the normal level of hydrogen sulfide in the brain is restored, oxidative and nitrative stress is inhibited, the further development of the disease process is blocked, and the therapeutic effect of hydrogen sulfide gas on central nervous system injury diseases is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the characterization results of four liposomes in Example 1, wherein, A is the transmission electron micrograph, B is the potential analysis diagram, and C is the particle size analysis diagram.

[0029] Figure 2 Cumulative release curves of RVG29 / TK@Lip / GYY and Lip / GYY in pH 7.4 phosphate buffer solution (PBS) with / without 100 μM H2O2.

[0030] Figure 3 In [figure], A is the expression of neuronal nitric oxide synthase (nNOS) in SH-SY5Y model cells treated with different dosing groups in Example 3, B is the semi-quantitative result of the expression of neuronal nitric oxide synthase in SH-SY5Y model cells treated with different dosing groups, and C is the statistical result of the nitric oxide content in SH-SY5Y model cells treated with different dosing groups.

[0031] Figure 4 Statistical results of the reactive oxygen species content in SH-SY5Y model cells treated with different dosing groups in Example 4.

[0032] Figure 5 Semi-quantitative map of the fluorescence intensity in the brain at the 4-hour time point of Parkinson's disease model mice treated with different dosing groups in Example 5.

[0033] Figure 6 Expression levels of tyrosine hydroxylase (TH) in Parkinson's disease model mice treated with different dosing groups in Example 6. Among them, A is the expression of tyrosine hydroxylase in the substantia nigra (SN) region, and B is the semi-quantitative result of the expression of tyrosine hydroxylase in the substantia nigra region.

[0034] Figure 7 Statistical charts of the behavior of Parkinson's disease model mice treated with different dosing groups in Example 7. Among them, A is the statistical chart of the total movement distance in the open field test, B is the statistical chart of the movement distance in the central area of the open field test, C is the statistical chart of the stationary time in the open field test, D is the statistical chart of the average speed in the open field test, E is the statistical chart of the turning and climbing pole time in the pole climbing test, and F is the statistical chart of the staying time on the rotating rod in the fatigue rotating rod test.

[0035] Figure 8 Expression levels of tyrosine hydroxylase (TH) and dopamine in Parkinson's disease model mice treated with different dosing groups in Example 8. Among them, A is the immunohistochemical staining of tyrosine hydroxylase in the substantia nigra (SN) and striatum (ST) regions, B is the expression of tyrosine hydroxylase in the substantia nigra and striatum regions, C is the semi-quantitative result of the expression of tyrosine hydroxylase in the substantia nigra region, D is the semi-quantitative result of the expression of tyrosine hydroxylase in the striatum region, E is the content of dopamine in the substantia nigra region, and F is the content of dopamine in the striatum region.

[0036] Figure 9 Map of the hydrogen sulfide content in the striatum region of Parkinson's disease model mice treated with different dosing groups in Example 9.

[0037] In the figure, the significance analysis result "ns" indicates no significant difference, "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. Detailed implementation manners

[0038] To make the objectives, features, and advantages of the present invention more obvious and understandable, the following provides a detailed description through specific implementation manners. Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined correspondingly without conflict.

[0039] For the operation methods without specific conditions noted in the following examples, they are generally in accordance with conventional conditions or the conditions recommended by the manufacturer. The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art. The experimental materials used in the following examples can be obtained from conventional biochemical reagent companies without special instructions.

[0040] It should be particularly noted that the selected control therapeutic drugs in the specific embodiments include drug-free blank liposomes RVG29 / TK@Lip, free drug hydrogen sulfide donor GYY, and non-brain-targeted and non-reactive oxygen-responsive drug-loaded liposomes Lip / GYY, to compare the therapeutic effect of brain-targeted and reactive oxygen-responsive hydrogen sulfide donor liposomes RVG29 / TK@Lip / GYY on Parkinson's disease (PD).

[0041] Example 1 Preparation and characterization of brain-targeted and reactive oxygen-responsive hydrogen sulfide donor liposomes

[0042] Liposomes were prepared by the thin-film dispersion method. 7.0 mg of distearoylphosphatidylcholine, 2.0 mg of cholesterol, and 2.0 mg of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 were accurately weighed into an eggplant-shaped flask, and added to a mixed system of 8.25 mL of dichloromethane and 2.75 mL of methanol. The organic solvents were removed by rotary evaporation. 11 mL of deionized water was added, and hydration was completed by stirring in a water bath at 37 °C for 30 min. Sonication was performed with a 400 W probe at 4 °C (40 cycles, 2 s on, 3 s off), and non-brain-targeted and non-reactive oxygen-responsive blank liposomes Lip were obtained after ultrafiltration and centrifugation. 1.0 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000-RVG29 and 1.0 mg of distearoylphosphatidylethanolamine-ketothiol-polyethylene glycol 2000 were used to replace 2.0 mg of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, and brain-targeted and reactive oxygen-responsive blank liposomes RVG29 / TK@Lip were prepared.

[0043] GYY and the lipid material were dissolved in dichloromethane and methanol at a mass ratio of 1:11. The brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposomes RVG29 / TK@Lip / GYY and the non-brain-targeted non-reactive oxygen species-responsive drug-loaded liposomes Lip / GYY were prepared in the same way.

[0044] The morphology, particle size, and zeta potential of Lip, RVG29 / TK@Lip, Lip / GYY, and RVG29 / TK@Lip / GYY were measured. The results are shown in A-C of Figure 1 The particle size of RVG29 / TK@Lip / GYY was about 105 nm and the zeta potential was about -23 mV.

[0045] The drug loading and encapsulation efficiency of the drug-loaded liposomes were determined by ultracentrifugation. The drug loading of RVG29 / TK@Lip / GYY was 3 wt% - 6 wt%, and the encapsulation efficiency was 45 wt% - 65 wt%.

[0046] Example 2 In vitro release study of brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposomes

[0047] Take 1.0 mg / mL of RVG29 / TK@Lip / GYY and Lip / GYY, and add H2O2 to make its final concentration 100 μM. At 0, 0.5 h, 1 h, 2 h, and 4 h, the volume-average particle size and particle size distribution were measured respectively. The results are shown in Table 1. When RVG29 / TK@Lip / GYY was co-incubated with H2O2 for 2 h, particle peaks at about 50 nm and 5000 nm appeared in the volume-average particle size distribution, indicating that the liposome structure had been damaged; when co-incubated with H2O2 for 4 h, particle peaks at about 5000 nm and 50 nm still appeared in the volume-average particle size distribution, and the percentage of the large particle peak at about 5000 nm increased significantly, indicating that the liposome structure had been completely damaged. The change in the volume-average particle size distribution of RVG29 / TK@Lip / GYY was consistent with the change in the volume-average particle size distribution, and the number-average particle size decreased to 45 nm. There was no obvious change in the volume-average, intensity-average, and number-average particle size distributions of Lip / GYY within 4 h in a 100 μM H2O2 environment. The above results indicate that RVG29 / TK@Lip / GYY has reactive oxygen species sensitivity, while Lip / GYY does not have reactive oxygen species sensitivity.

[0048] Table 1 Volume-average particle size and its distribution percentage of RVG29 / TK@Lip / GYY in a 100 μM H2O2 environment

[0049]

[0050] Using GYY as the object to be measured, and PBS solution with pH 7.4 containing or not containing 100 μM H2O2 as the release medium to ensure that the drug release situation meets the sink conditions. Take Lip / GYY and RVG29 / TK@Lip / GYY liposome solutions, place them in dialysis bags (MWCO = 3.5 kDa) respectively, oscillate in a 37°C constant temperature water bath (100 rpm), sample at 0, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 24 h in turn, and replace with fresh release medium after sampling. According to the methylene blue method, use a hydrogen sulfide test kit to measure the concentration of GYY and calculate the cumulative release percentage of GYY. The results are as Figure 2 shown. Compared with the normal release conditions, for the reactive oxygen species-responsive drug-loaded liposome RVG29 / TK@Lip / GYY, the cumulative release percentage of GYY at the 8 h time point increased from 51.1% in the release medium without H2O2 to 82.2% in the release medium containing H2O2, and the cumulative release percentage of GYY at the 24 h time point increased from 64.2% in the release medium without H2O2 to 88.1% in the release medium containing H2O2. For the non-reactive oxygen species-responsive liposome Lip / GYY, the cumulative release percentage of GYY at the 8 h time point in the release medium containing H2O2 was 55.1%, and the cumulative release percentage of GYY at the 24 h time point was 70.2%. The results indicate that the reactive oxygen species-sensitive drug-loaded liposome has good sensitive release characteristics in the in vitro simulated reactive oxygen species environment.

[0051] Example 3 In vitro anti-nitration effect of brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome

[0052] Take SH-SY5Y cells in good growth state, inoculate them in a 6-well plate at a density of 2.0×10 5 cells / well, culture at 37°C and 5% CO2 until the cells adhere to the wall. When the cell confluence reaches 75%, add RVG29 / TK@Lip, GYY, Lip / GYY or RVG29 / TK@Lip / GYY drug-loaded liposome (containing 100 μM GYY), pre-incubate with SH-SY5Y cells for 1 h, and then add 500 μM N-methyl-4-phenylpyridinium iodide (MPP + ), and continue to co-incubate for 24 h. Discard the culture medium, rinse 3 times with pre-cooled PBS, extract proteins with a protein lysis solution at 4°C. Collect the cell suspension, centrifuge at 13,000 rpm for 15 minutes at 4°C using a high-speed centrifuge, take the supernatant, detect the total protein concentration, and dilute to the same concentration with deionized water. Boil the protein solution at 100°C for 10 min for denaturation. Perform Western blot analysis, and the results are as Figure 3 shown in A - B of

[0053] Take SH-SY5Y cells in good growth state, at a density of 8.0×10 3The density of cells per well was inoculated into a black-bottom transparent 96-well plate and cultured at 37 °C and 5% CO2 until the cells adhered. When the cell confluence reached 75%, RVG29 / TK@Lip, GYY, Lip / GYY or RVG29 / TK@Lip / GYY drug-loaded liposomes (containing 100 μM GYY) were added and pre-incubated with SH-SY5Y cells for 1 h. Subsequently, 500 μM N-methyl-4-phenylpyridinium iodide (MPP + ) was added, and the co-incubation was continued for 24 h. The culture medium was discarded, and the DAF-FM-DA fluorescent probe was diluted to the working solution concentration (containing 5 μM DAF-FM-DA) with serum-free medium at a ratio of 1:1000 and added to the above cells. The cells were incubated at 37 °C and 5% CO2 in the dark for 30 min. The culture medium was discarded, and the cells were rinsed 3 times with PBS. The fluorescence intensity of each group of cells at 488 nm was detected by a fluorescence microplate reader. The results are as shown in Figure 3 C in

[0054] According to Figure 3 the results in A-C in + , compared with the Ctrl group, the expression of neuronal nitric oxide synthase (nNOS) in SH-SY5Y cells was significantly up-regulated after MPP + treatment, and the content of nitric oxide in living cells increased significantly. After drug treatment, the expression of nNOS was down-regulated; compared with the GYY group, the RVG29 / TK@Lip / GYY group significantly reduced the expression of nNOS and restored it to the normal physiological level. Compared with the MPP + group, the content of nitric oxide in the GYY group, Lip / GYY group and RVG29 / TK@Lip / GYY group decreased, and the fluorescence intensity decreased by 12.6%, 14.6% and 27.6% respectively. Among them, RVG29 / TK@Lip / GYY maximally scavenged intracellular NO and restored it to the normal physiological level.

[0055] Example 4 Antioxidant Ability of Brain-Targeted Reactive Oxygen Species-Responsive Hydrogen Sulfide Donor Liposomes

[0056] SH-SY5Y cells in good growth state were taken and inoculated into a black-bottom transparent 96-well plate at a density of 8×10 3 cells per well and cultured at 37 °C and 5% CO2 until the cells adhered. When the cell confluence reached 75%, RVG29 / TK@Lip, GYY, Lip / GYY or RVG29 / TK@Lip / GYY drug-loaded liposomes (containing 100 μM GYY) were added and pre-incubated with SH-SY5Y cells for 1 h. Subsequently, 500 μM MPP +, Incubate for another 24 h. Discard the medium, dilute the reactive oxygen species fluorescent probe DCFH-DA to the working solution concentration (containing 10 μM DCFH-DA) with serum-free medium at a ratio of 1:1000, add it to the above cells, incubate at 37 °C and 5% CO2 in the dark for 30 min, discard the medium, wash 3 times with PBS, and detect the fluorescence intensity of each group of cells at 488 nm with a fluorescence microplate reader. The results are as Figure 4 shown, MPP + group, GYY group, Lip / GYY group, and RVG29 / TK@Lip / GYY group were 1.75-fold, 1.55-fold, 1.41-fold, and 1.10-fold that of the Ctrl group in terms of intracellular ROS fluorescence intensity, respectively, indicating that the RVG29 / TK@Lip / GYY group had the greatest reduction in fluorescence intensity. The research results show that RVG29 / TK@Lip / GYY can effectively scavenge intracellular ROS and inhibit oxidative stress.

[0057] Example 5 Brain Distribution of Brain-Targeted Reactive Oxygen Species-Responsive Hydrogen Sulfide Donor Liposomes

[0058] Using C57BL / 6 mice as model animals, weigh the mice and intraperitoneally inject 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 16 mg / kg, inject once every 2 h intraperitoneally, for a total of 4 injections, to construct an acute PD mouse model.

[0059] Weigh 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and dissolve it in absolute ethanol. Prepare Lip / DiR and RVG29 / TK@Lip / DiR by the thin film dispersion method in Example 1. According to the dose of 0.5 mg / kg DiR, inject Lip / DiR and RVG29 / TK@Lip / DiR via the tail vein, and observe the brain distribution of Lip / DiR and RVG29 / TK@Lip / DiR with a small animal in vivo imager at 2 h, 4 h, 8 h, 12 h, and 24 h.

[0060] Figure 5 It shows that the fluorescence signal in the brains of mice in the RVG29 / TK@Lip / DiR group was significantly stronger than that in the Lip / DiR group at the 4 h time point, and the fluorescence signal intensity of the RVG29 / TK@Lip / DiR group was 1.53 times that of the Lip / DiR group, indicating that the modification of RVG29 significantly promoted the transport of liposomes across the BBB and enhanced the brain targeting ability of RVG29 / TK@Lip / DiR.

[0061] Example 6: Study on the Restoration of Tyrosine Hydroxylase Level in PD Model Animals by Brain-Targeted Reactive Oxygen Species-Responsive Hydrogen Sulfide Donor Liposomes at Different Doses

[0062] Set up the Ctrl group, PD group, GYY 65 μM / kg group, GYY 130 μM / kg group, RVG29 / TK@Lip / GYY 65 μM / kg group, and RVG29 / TK@Lip / GYY 130 μM / kg group. Before inducing the acute PD model mice with MPTP, each group was pre-treated for 3 days, once a day at the same time. The specific protocol is as follows: Mice in the Ctrl group and PD group were injected with normal saline via the tail vein, and mice in the other four groups were injected with the corresponding drugs or preparations via the tail vein. On the 4th day, acute PD model mice were constructed. The mice were weighed and intraperitoneally injected with MPTP at 16 mg / kg, once every 2 hours for a total of 4 injections. At the same time, the Ctrl group was given an equal amount of normal saline. After inducing the acute PD model mice with MPTP, each group of mice was continuously injected with normal saline, the corresponding drugs or preparations via the tail vein for 7 days.

[0063] After the treatment was completed, mice were randomly selected from each group, anesthetized and sacrificed, and the whole brain was taken. The residual bloodstains on the brain surface were washed with PBS, and the substantia nigra was isolated on ice, homogenized, and proteins were extracted with protein lysate. The changes in tyrosine hydroxylase (TH) in the substantia nigra region of each experimental group were analyzed by Western blotting. The results are shown in Figure 6 A - B in

[0064] According to Figure 6 the results in A - B in

[0065] Example 7: Brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposomes improve PD motor symptoms

[0066] The Ctrl group, PD group, RVG29 / TK@Lip group, GYY group, Lip / GYY group, and RVG29 / TK@Lip / GYY group were set up. Before constructing the acute PD mouse model induced by MPTP, each group was pre-treated for 3 days, once a day at the same time, and the specific protocol was as follows: Mice in the Ctrl group and PD group were injected with normal saline via the tail vein, and mice in the other four groups were injected with the corresponding drugs or preparations (calculated based on the concentration of GYY, 130 μM / kg) via the tail vein. On the 4th day, acute PD model mice were constructed. The mice were weighed and intraperitoneally injected with MPTP at 16 mg / kg, once every 2 hours for a total of 4 injections, and the Ctrl group was given an equal amount of normal saline at the same time. After constructing the acute PD mouse model induced by MPTP, each group of mice was injected with normal saline, the corresponding drugs or preparations via the tail vein for continuous treatment for 7 days. After the treatment, behavioral training and testing were performed on each group of mice. After the behavioral testing, the brain tissues of each group of mice were taken for biochemical experiments and immunological experiments.

[0067] The open-field experiment was used to evaluate the changes in the spontaneous locomotor behavior and anxiety-like behavior of mice in each experimental group. The open-field experimental box was an acrylic cube of 45 cm × 45 cm × 45 cm. The Any-maze software was used to divide the activity area of the mice into a central area and a total area. After the experimental device was installed, the mice were placed in the central area of the open-field experimental box, and the experimenter quickly left. The mice freely explored and moved in the open-field experimental box for 5 minutes. The movement trajectory of the mice was recorded, and the total movement distance ( Figure 7 A in Figure 7 ), the movement distance in the central area ( Figure 7 B in Figure 7 ), the stationary time ( C in

[0068] ), and the average speed ( Figure 7 D in

[0069] Figure 7 F in

[0070] Figure 7 The pole climbing experiment was used to evaluate the motor coordination ability and motor tolerance of mice in each experimental group. The mice were placed on the top of a pole with a height of 60 cm, a diameter of 1 cm, an angle of 90°, a rough surface, and a circular cross-section. The time for the mice to turn their heads down and climb from the top to the bottom was recorded, and the results were as shown in Figure 7 E in

[0069] The fatigue rotarod experiment was used to evaluate the motor coordination and balance ability of mice in each experimental group. The time for the mice to move on the rotarod in the uniformly accelerating mode (from 4 rpm to 40 rpm within 300 s) was recorded, and the results were as shown in Figure 7 F in

[0070] Figure 7The results of A-F in [the relevant content] showed that, compared with the PD group and the blank liposome RVG29 / TK@Lip group, after treatment with the GYY group, Lip / GYY group, and RVG29 / TK@Lip / GYY group, the motor coordination ability, balance ability, and exercise endurance of PD model animals could be significantly improved, and the motor dysfunction and anxiety-like behavior changes of PD model mice could be alleviated. Moreover, the RVG29 / TK@Lip / GYY group had the best therapeutic effect, which was significantly better than the GYY group and the Lip / GYY group.

[0071] Example 8: Study on the restoration of tyrosine hydroxylase and dopamine expression levels in PD model animals by brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposomes

[0072] After the treatment ended according to Example 7, mice in each group were randomly selected, anesthetized and sacrificed, the whole brain was taken, the blood stains remaining on the brain surface were washed with PBS, fixed overnight with 4% paraformaldehyde, embedded in paraffin and sectioned, and immunohistochemical staining was used to observe the TH expression in the substantia nigra and striatum regions of each experimental group. The results were as shown in Figure 8 A in [the relevant content].

[0073] After the treatment ended according to Example 7, mice in each group were randomly selected, anesthetized and sacrificed, the whole brain was taken, the blood stains remaining on the brain surface were washed with PBS, the substantia nigra and striatum were isolated on ice, homogenized, and proteins were extracted with protein lysate. Western blotting was used to analyze the changes in TH in the substantia nigra and striatum regions of each experimental group. The results were as shown in Figure 8 B-D in [the relevant content]. The dopamine content in the substantia nigra and striatum regions of each experimental group was measured using a dopamine kit. The results were as shown in Figure 8 E-F in [the relevant content].

[0074] According to Figure 8 the results of A-F in [the relevant content], it could be known that the TH protein expression levels in the substantia nigra and striatum regions of the PD group were significantly lower than those of the Ctrl group, the TH immunohistochemical staining was the lightest, and the dopamine content was significantly decreased. The blank vector group had no therapeutic effect, and its TH expression level, immunohistochemical staining results, and dopamine content were all close to those of the PD group. After treatment with the GYY group and the Lip / GYY group, the TH protein expression level and dopamine content increased respectively, the TH immunohistochemical staining became darker, and the RVG29 / TK@Lip / GYY group further improved the therapeutic effect, with the deepest TH immunohistochemical staining, significantly enhancing the efficacy of GYY and promoting the expression of TH protein and the production of dopamine.

[0075] Example 9: Study on the restoration of hydrogen sulfide content in the brain of PD model animals by brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposomes

[0076] After the treatment according to Example 7, mice in each group were randomly selected, anesthetized and sacrificed, and their whole brains were taken. The bloodstains on the surface were washed with PBS, and the striatum was isolated on ice. The hydrogen sulfide content in the striatum of mice in each group was measured using a hydrogen sulfide kit. The results are as follows Figure 9 shown. Compared with the Ctrl group, the hydrogen sulfide content in the PD group and the blank liposome RVG29 / TK@Lip group was significantly decreased, and the hydrogen sulfide content in the GYY group and the Lip / GYY group was not significantly increased. The hydrogen sulfide content in the RVG29 / TK@Lip / GYY group was basically restored to the level of the Ctrl group.

[0077] The embodiments described above have elaborated on the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and do not limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the principle scope of the present invention shall be included within the protection scope of the present invention.

Claims

1. A brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome, characterized in that, The structure includes a lipid material and a hydrogen sulfide donor encapsulated within the lipid material. The lipid material includes phospholipids, cholesterol, reactive oxygen species (ROS)-sensitive lipids, and brain-targeting lipids. The ROS-sensitive lipids include a phospholipid lipophilic moiety, a polyethylene glycol hydrophilic moiety, and a ROS-sensitive bond. The brain-targeting lipids include a phospholipid lipophilic moiety, a polyethylene glycol hydrophilic moiety, and a brain-targeting peptide.

2. The brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 1, wherein The hydrogen sulfide donor is GYY4137.

3. The brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 1, wherein The phospholipids are selected from distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, or phosphatidylcholine; the ROS-sensitive bond in the ROS-sensitive lipids is a ketothiol bond; the brain-targeting peptide in the brain-targeting lipids is the RVG29 peptide.

4. The brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 1, wherein The ROS-sensitive lipids are selected from distearoylphosphatidylethanolamine-ketothiol-polyethylene glycol, and the brain-targeting lipids are selected from distearoylphosphatidylethanolamine-polyethylene glycol-RVG29.

5. The brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 1, wherein The particle size of the brain-targeting ROS-responsive hydrogen sulfide donor liposomes is ≤300 nm, and the drug loading is 3 wt% - 6 wt%.

6. The preparation method of the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to any one of claims 1-5, characterized in that, Using the thin film dispersion method, it specifically includes the following steps: S01 Dissolve the hydrogen sulfide donor and the lipid material in an organic solvent to obtain an organic phase; S02 Rotavaporize the organic phase under heating conditions to obtain an organic phase film. Add water to the organic phase film, hydrate, sonicate, and ultrafiltrate and centrifuge to obtain the brain-targeting ROS-responsive hydrogen sulfide donor liposomes.

7. The preparation method of the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 6, characterized in that, The organic solvent is a mixed solution of dichloromethane and methanol.

8. The preparation method of the brain-targeted reactive oxygen species-responsive hydrogen sulfide donor liposome according to claim 6, characterized in that, In step S01, the feeding mass ratio of the hydrogen sulfide donor to the lipid material is 0.5 - 2:11; in the lipid material, the feeding mass ratio of phospholipids, cholesterol, ROS-sensitive lipids, and brain-targeting lipids is 6 - 8:1 - 3:1:

1.

9. A therapeutic drug for central nervous system injury diseases, characterized in that, It includes the brain-targeting ROS-responsive hydrogen sulfide donor liposomes according to any one of claims 1 - 5.

10. The therapeutic drug for central nervous system injury diseases according to claim 9, characterized in that, The administration method of the drug for treating central nervous system injury diseases is intravenous injection.

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