A thylakoid-like composite vesicle and a preparation method and application thereof

Abstract

The application provides a kind of thylakoid composite vesicle and its preparation method and application.The thylakoid composite vesicle is formed by mixing target liposome and thylakoid membrane fragments according to the mass ratio of 100:1-5;The target liposome is formed by mixing neutral lipid, steroidal lipid, brain targeting modifier containing RVG29 peptide and REV-ERB alpha / beta agonist according to the mass ratio of (7-12):1:(0.2-4):(0.82-1.7).The composite vesicle of the application realizes active targeted penetration of blood-brain barrier through RVG29 polypeptide, and regulates single node of molecular clock through REV-ERB alpha / beta agonist, and carries QPRT of thylakoid membrane to catalyze in situ synthesis of NAD + The rhythm mediated global regulation is coordinated to realize comprehensive and accurate intervention on IL-17 secretion rhythm of gamma delta T cells.

Description

Technical Field

[0001] This invention belongs to the field of bio-industry, especially the field of medical nanoparticle formulation technology, specifically relating to a vesicle-like composite vesicle, its preparation method and its application in the subacute treatment of ischemic stroke. Background Technology

[0002] Ischemic stroke is one of the leading causes of death and long-term disability worldwide. Currently, intravenous thrombolysis and endovascular thrombectomy are the main treatments in the acute phase, but effective pharmacological intervention strategies are lacking for patients in the subacute phase. In the subacute phase, the continued progression of neuroinflammation is the core pathological mechanism leading to secondary brain injury and neurological deterioration. Among these mechanisms, IL-17, secreted by γδT cells, is a key pro-inflammatory cytokine, and its secretion level exhibits a significant diurnal rhythm. After stroke, the molecular clock system of these cells is disrupted, leading to abnormal IL-17 secretion rhythm, thereby exacerbating neuroinflammatory damage and hindering neurological function recovery.

[0003] The molecular clock system maintains precise regulation of circadian rhythms through the transcription-translation feedback loop (TTFL). Existing small molecule drugs for rhythm regulation (such as SR9009) mainly act on a single node of the molecular clock feedback loop (such as REV-ERBα), and can only regulate a limited number of parameters such as the median level of the rhythm. They cannot synchronously coordinate the multidimensional dynamic characteristics of the TTFL, and therefore cannot fully restore the normal rhythmic phenotype of immune cells, thus limiting their clinical translational applications.

[0004] Nicotinamide adenine dinucleotide (NAD) + As a key molecule in energy metabolism and a core coenzyme of the molecular clock system, BMAL1 plays a pivotal role in regulating core rhythmic parameters such as amplitude, period, and phase of the molecular clock by activating Sirt1 to mediate the deacetylation modification of BMAL1. Following stroke, NAD3 in brain tissue... + The levels of BMAL1 decreased significantly, Sirt1 activity was inhibited, and BMAL1 acetylation levels increased, leading to "NAD" + A vicious cycle of "exhaustion - molecular clock disruption - amplified inflammation" exists. Current methods to increase NAD+... + The strategy of treatment mainly relies on exogenous supplementation of precursor substances (such as nicotinamide mononucleotide NMN and nicotinamide riboside NR). These substances have low conversion efficiency, and long-term supplementation can easily lead to metabolic dependence, resulting in limited therapeutic effects.

[0005] In recent years, plant-derived thylakoid membranes have demonstrated excellent biocompatibility and membrane fusion capabilities as novel bionanocarriers. Notably, during thylakoid membrane extraction, quinolinate phosphoribosyltransferase (QPRT) is released from the chloroplast stroma and co-precipitates with thylakoid membrane fragments. Therefore, thylakoid membrane fragments typically carry QPRT (https: / / doi.org / 10.1038 / s41551-025-01402-y). QPRT can catalyze the de novo synthesis of quinolinate, leading to NAD+. + Quinolinate is generated autonomously in situ. In the ischemic microenvironment following stroke, the tryptophan-kynurenine metabolic pathway is overactivated, leading to a large accumulation of quinolinate and neurotoxicity. Utilizing the QPRT enzyme activity carried by thylakoid membrane fragments, pathologically accumulated quinolinate can be effectively consumed, thereby driving NAD+ metabolism. + NAD supplementation via de novo synthesis pathway + Repair NAD + -Sirt1-BMAL1 axis function, thereby comprehensively reshaping the IL-17 secretion rhythm of γδT cells. However, the thylakoid membrane itself lacks the ability to penetrate the blood-brain barrier.

[0006] Therefore, there is an urgent need to develop a nanomedicine that can penetrate the blood-brain barrier for targeted delivery and precisely and comprehensively regulate the IL-17 secretion rhythm of γδT cells, so as to achieve precise intervention on the immune rhythm disorder in the subacute phase of ischemic stroke. Summary of the Invention

[0007] The purpose of this invention is to provide a thylakoid complex vesicle for immune rhythm regulation in the subacute phase of ischemic stroke and its preparation method, aiming to solve the following technical problems existing in the prior art: (1) There is a lack of effective immune rhythm intervention methods in the subacute phase of ischemic stroke; (2) Existing rhythm regulation drugs can only act on a single node of the molecular clock feedback loop, and cannot synchronously coordinate the multidimensional dynamic characteristics of TTFL, making it difficult to fully restore the normal rhythmic phenotype of immune cells; (3) Existing drugs that enhance NAD + The strategy of level mainly relies on the supplementation of exogenous precursors. Long-term supplementation is prone to metabolic dependence and cannot accurately target immune cells with disordered rhythms; (4) Plant thylakoid membranes alone lack the ability to penetrate the blood-brain barrier and the function of regulating the median rhythm.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a targeted liposome composed of neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in a mass ratio of (7-12):1:(0.2-4):(0.82-1.7).

[0010] In the preferred targeted liposomes of the present invention, the neutral lipid may be selected from any one or a mixture of two of phosphatidylethanolamine or phosphatidylcholine; phosphatidylcholine is further preferred; and soybean lecithin is most preferred.

[0011] In the preferred targeted liposomes of the present invention, the steroidal lipids may be selected from any one or a mixture of two or more of phytosterols such as β-sitosterol or cholesterol; cholesterol is further preferred.

[0012] In a preferred embodiment of the present invention, the brain-targeting modifier containing RVG29 peptide is a conjugate containing RVG29 peptide and membrane-anchored lipid groups; in a further preferred embodiment, the brain-targeting modifier containing RVG29 peptide is a conjugate formed by linking RVG29 peptide and membrane-anchored lipid groups via a hydrophilic linker arm.

[0013] In the preferred targeted liposomes of the present invention, the REV-ERBα / β agonist can be either SR9011 or SR9009; SR9009 is the most preferred.

[0014] In a preferred embodiment of the present invention, the targeted liposome is composed of a mixture of neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in a mass ratio of (7-10):1:(1-4):(0.9-1.4); more preferably, it is composed of a mixture of neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in a mass ratio of 7:1:(2-4):(1-1.2); most preferably, it is composed of a mixture of neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in a mass ratio of 7:1:4:1.2.

[0015] The targeted liposomes described in this invention can be prepared using conventional liposome preparation methods. For example, they can be prepared according to the following steps:

[0016] Neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and REV-ERBα / β agonists were dissolved in an organic solvent at a mass ratio of (7-12):1:(0.2-4):(0.82-1.7). The mixture was then placed in a rotary evaporator and evaporated under reduced pressure at 37°C to remove the organic solvent, forming a uniform lipid film on the inner wall of the container. Water was then added, and the mixture was dispersed by ultrasonic oscillation in an ice-water bath to obtain targeted liposomes.

[0017] In a preferred embodiment of the present invention, the mass ratio of neutral lipids, steroidal lipids, brain-targeting modifiers containing RVG29 peptide, and REV-ERBα / β agonists in the preparation method is (7-10):1:(1-4):(0.9-1.4), more preferably 7:1:(2-4):(1-1.2), and most preferably 7:1:4:1.2. This most preferred mass ratio range ensures the effective loading of RVG29 peptides on the liposome surface while achieving the maximum encapsulation efficiency of REV-ERBα / β agonists.

[0018] The targeted liposomes of this invention are endowed with blood-brain barrier targeting and penetration capabilities through surface-modified RVG29 peptides, which can effectively downregulate the median level of IL-17 secretion by γδT cells.

[0019] In a second aspect, the present invention provides a thylakoid composite vesicle, which is composed of the targeted liposomes and thylakoid membrane fragments described in the first aspect of the present invention mixed at a mass ratio of 100:1-5, with a particle size of 80-150 nm; the mass of the thylakoid membrane fragments is based on the mass of chlorophyll they contain.

[0020] In the preferred composite vesicles of this invention, the thylakoid membrane fragments are derived from any one of the QPRT-rich plants such as spinach, lettuce, and cabbage; more preferably from spinach, which has the optimal QPRT content.

[0021] In the composite vesicles described in this invention, the mass ratio of the targeting liposomes to the thylakoid membrane fragments has a significant impact on delivery efficiency and immune rhythm regulation. Excessive thylakoid membrane content leads to a lower proportion of targeting liposomes, thus affecting the RVG29 peptide content on the composite vesicle surface and weakening its ability to target cells. Conversely, excessively low thylakoid membrane content results in insufficient QPRT enzyme activity. Therefore, in the preferred composite vesicles of this invention, the mass ratio of the targeting liposomes to the thylakoid membrane fragments is 40:1-1.5. In the most preferred composite vesicles of this invention, the mass ratio of the targeting liposomes to the thylakoid membrane fragments is 40:1. At this ratio, the composite vesicles can maximize the thylakoid membrane loading while maintaining the blood-brain barrier penetration effect of the RVG29 targeting peptide, ensuring the effective exertion of QPRT enzyme activity.

[0022] Thirdly, the present invention provides a method for preparing the thylakoid composite vesicles described in the second aspect, comprising:

[0023] 1) Extract thylakoid membrane fragments from the leaves of plants rich in QPRT;

[0024] 2) Neutral lipids, steroidal lipids, brain-targeting modifiers containing RVG29 peptide, and REV-ERBα / β agonists were dissolved in an organic solvent at a mass ratio of (7-12):1:(0.2-4):(0.82-1.7) to prepare targeted liposomes;

[0025] 3) The thylakoid membrane fragments obtained in 1) and the targeted liposomes obtained in 2) were blended and extruded at a mass ratio of 1-5:100 to obtain thylakoid composite vesicles with a particle size of 80-150 nm; wherein the amount of thylakoid membrane fragments fed was quantified based on chlorophyll content.

[0026] In the preparation method of the present invention, 1) the QPRT-rich plant is selected from any one or a mixture of two or more of spinach, lettuce or cabbage; spinach is preferred.

[0027] In the preparation method described in this invention, 1) the method for extracting thylakoid membrane fragments is not particularly limited and can be any existing extraction method, with the following methods being preferred:

[0028] Take fresh spinach, lettuce, or cabbage leaves, wash them, and store them overnight in the dark at 4°C. Place the leaves in pre-cooled homogenizing buffer and homogenize them using a high-speed homogenizer. Filter the resulting homogenate through a 70 μm cell sieve, collect the filtrate, and centrifuge at 8000 rpm for 10 min. Discard the supernatant and collect the precipitate. Gently resuspend the precipitate in hypotonic lysis buffer and allow it to lyse for 2 h. Then centrifuge the lysis buffer at 8000 rpm for 10 min and collect the precipitate containing thylakoid membrane fragments. Wash the precipitate twice with 10 mM HEPES buffer and resuspend it in osmotic shock buffer.

[0029] In the preparation method described in this invention, the definitions of neutral lipids, steroidal lipids, brain-targeting modifiers containing RVG29 peptide, and REV-ERBα / β agonists in 2) are the same as those in the first aspect of this invention, and will not be repeated here.

[0030] In the preparation method described in this invention, the preferred mass ratio of the neutral lipids, steroidal lipids, brain-targeting modifiers containing RVG29 peptide, and REV-ERBα / β agonists in step 2) is (7-10):1:(1-4):(0.9-1.4), more preferably 7:1:(2-4):(1-1.2), and most preferably 7:1:4:1.2. This optimal ratio range ensures the effective loading of RVG29 peptides on the liposome surface while achieving maximum encapsulation efficiency of the REV-ERBα / β agonist.

[0031] In the preparation method of the present invention, the preferred mass ratio of the targeted liposomes and thylakoid membrane fragments in 3) is 100:2.5. This mass ratio can maximize the loading of the thylakoid membrane while maintaining the blood-brain barrier penetration effect of the RVG29 targeted peptide, thus ensuring the effective performance of QPRT enzyme activity.

[0032] In the preparation method described in this invention, the particle size of the thylakoid composite vesicles obtained in step 3) is preferably 100 nm.

[0033] Fourthly, the present invention provides the application of the thylakoid complex vesicles described in the second aspect of the present invention, or the thylakoid complex vesicles prepared by the method described in the third aspect of the present invention, in the preparation of drugs for the subacute phase treatment of ischemic stroke.

[0034] In the application described in this invention, the therapeutic drug can be targeted and delivered to the cerebral ischemic microenvironment via intravenous injection to synergistically regulate the multidimensional rhythm parameters of IL-17 secretion by γδT cells, thereby achieving a comprehensive remodeling of immune rhythm homeostasis.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] (1) Excellent biosafety:

[0037] This invention uses plant-derived thylakoid membranes and biocompatible phospholipid materials to construct composite vesicles, which are free from hepatotoxicity and nephrotoxicity and have good biocompatibility.

[0038] (2) Comprehensive and precise reshaping of immune rhythm:

[0039] The composite vesicles described in this invention achieve active targeted penetration of the blood-brain barrier via the RVG29 peptide, and regulate a single node of the molecular clock through REV-ERBα / β agonists such as SR9009, thereby catalyzing the in-situ synthesis of NAD from pathologically accumulated quinolinic acid using QPRT contained in the thylakoid membrane fragments. + The mediated global regulation of rhythm works in synergy to achieve comprehensive and precise intervention on the IL-17 secretion rhythm of γδT cells. Attached Figure Description

[0040] Figure 1 This is a transmission electron microscope image of the targeted liposomes prepared in Example 1.

[0041] Figure 2 This is a transmission electron microscope image of the thylakoid composite vesicles prepared in Example 2.

[0042] Figure 3 This reflects the characterization of the particle size and potential of the four vesicles in Experiment Example 1. Part (A) reflects the hydration dynamics particle size of the four vesicles; Part (B) reflects the potential of the four vesicles.

[0043] Figure 4 This reflects the endocytosis flow cytometry results of the four types of vesicles in Experiment Example 2. Part (A) shows the Dio fluorescence detection results of the four types of vesicles; Part (B) shows the thylakoid (TK) fluorescence detection results of the four types of vesicles.

[0044] Figure 5 This is a liquid chromatogram of the four vesicles loaded with SR9009 in Experiment Example 3.

[0045] Figure 6 These are the gel electrophoresis images and Western blot images of the five samples in Experiment Example 4; where: (A) is the gel electrophoresis image; (B) is the Western blot image.

[0046] Figure 7 This demonstrates the targeting of different thylakoid complex vesicles to the brain tissue of mice with ischemic stroke in Experiment Example 5. Part (A) shows near-infrared fluorescence imaging images of brain tissue from three parallel samples of mice 24 hours after tail vein injection of each group of thylakoid complex vesicles; Part (B) shows the quantitative fluorescence results of mouse brain tissue at different time intervals after tail vein injection of each group of thylakoid complex vesicles.

[0047] Figure 8 This illustrates the regulation of IL-17 secretion rhythm by different thylakoid complex vesicles in mice with ischemic stroke in Experiment Example 5. Specifically: (A) shows the rhythm curves of IL-17 secretion by γδT cells in the brain tissue of healthy and diseased mice; (B) shows the rhythm curves of IL-17 secretion by γδT cells in the brain tissue of different groups of mice after 5 days of treatment.

[0048] Figure 9 This reflects the TTC staining results of brain tissue from different groups of mice 7 days after being treated with different thylakoid complex vesicles in Experiment Example 5.

[0049] Figure 10 This illustrates the NAD5 levels in the brain tissue of mice from different groups after 7 days of treatment with different drugs for ischemic stroke in Experiment 5. + level.

[0050] Figure 11 This reflects the results of liver injury marker detection in mouse blood 7 days after tail vein injection of the thylakoid composite vesicles prepared in Example 2 in Experiment 5. Part (A) shows the alanine aminotransferase detection results, part (B) shows the aspartate aminotransferase detection results, and part (C) shows the creatinine content detection results. Detailed Implementation

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following specific examples are only used to explain this application and do not represent or limit the scope of protection of the present invention.

[0052] Example 1. Preparation of brain-targeting liposomes

[0053] This embodiment provides a brain-targeting liposome, the preparation steps of which are as follows:

[0054] Soybean lecithin, cholesterol, DSPE-PEG-RVG29, and SR9009 were dissolved in chloroform at a mass ratio of 7:1:4:1.2. The solution was then evaporated under reduced pressure at 37°C in a rotary evaporator to remove the organic solvent, forming a uniform lipid film on the inner wall of the container. Water was then added, and the mixture was dispersed by ultrasonic oscillation in an ice-water bath to obtain brain-targeting liposomes, designated "Lip-RVG29@SR9009". Their microstructure is shown below. Figure 1 The transmission electron microscope image shows that the liposome particle size is no more than 100 nm.

[0055] Example 2. Preparation of thylakoid complex vesicles

[0056] This embodiment provides a vesicle-like composite vesicle, the preparation steps of which are as follows:

[0057] 1. Extraction and quantification of thylakoids, the steps are as follows:

[0058] Fresh spinach leaves were collected, washed, and stored overnight at 4°C in the dark. The leaves were homogenized using a high-speed homogenizer in a freezing buffer (20 mM Methyline, 0.4 M sucrose, 2 mM MgCl2, 40 mM NaCl, 0.2% bovine serum albumin (BSA), pH 7.8). The resulting solution was then filtered through a 70 μm filter, and the filtrate was collected at 8,000 rpm for 10 min. The precipitate was gently resuspended in hypotonic lysis buffer (10 mM HEPES, pH 8) for 2 h. The filtrate was then centrifuged at 8,000 rpm for 10 min, and the precipitate containing thylakoid membrane fragments was collected. The precipitate was washed twice with 10 mM HEPES buffer and then resuspended in osmotic shock buffer (20 mM MES, 5 mM MgCl2, 15 mM NaCl, 0.4 M sucrose, pH 6.5). The thylakoid membrane fragments were stored at -80°C until use. Before use, thylakoid membrane fragments should be thawed on ice and washed two to three times in HEPES buffer.

[0059] To quantify the thylakoid membrane fragments, 10 μL of the obtained thylakoid membrane fragment suspension was first mixed with 1,990 μL of acetone, and then centrifuged at 13,000 g for 2 minutes at 4 °C. The resulting chlorophyll-containing supernatant was characterized using a UV spectrophotometer, yielding light absorption intensities at peaks of 663 nm and 645 nm. The chlorophyll concentration was calculated using the following formula:

[0060] Total chlorophyll concentration (CT, μg / mL) = [20.31 × A] 645 + 8.05×A 663 ]×g

[0061] Where CT is the total chlorophyll concentration, CT = Ca + Cb = [20.31 × A] 645 +8.05×A 663 ×g; Ca is the concentration of Chl a, Cb is the concentration of Chl b, A 663 It is the light absorption intensity at the 663 nm peak, A 645 is the light absorption intensity at the 645 nm peak, and g is the dilution factor of the thylakoid membrane fragment suspension to acetone.

[0062] 2. Preparation and characterization of thylakoid complex vesicles, the steps of which are as follows:

[0063] The brain-targeting liposomes obtained in Example 1 were blended with the thylakoid membrane fragments obtained in Step 1 of this Example at a mass ratio of 1:0.025. The mixture was repeatedly extruded through a liposome extruder via a polycarbonate membrane with a pore size of 100 nm to obtain thylakoid composite vesicles, designated "Lip-TK-RVG29@SR9009-1". A transmission electron microscope image of the thylakoid composite vesicles prepared in this Example is shown below. Figure 2 This shows that the thylakoid composite vesicles in this embodiment have a uniform particle size of approximately 100 nm.

[0064] Example 3. Preparation of thylakoid composite vesicles

[0065] This embodiment provides a thylakoid composite vesicle, the preparation steps of which are largely the same as those in Example 2, except that in step 2, the brain-targeting liposomes obtained in Example 1 and the thylakoid membrane fragments obtained in step 1 are blended at a mass ratio of 1:0.01, and repeatedly extruded through a polycarbonate membrane with a pore size of 100 nm using a liposome extruder to obtain the thylakoid composite vesicle, denoted as "Lip-TK-RVG29@SR9009-2".

[0066] Example 4. Preparation of thylakoid complex vesicles

[0067] This embodiment provides a thylakoid composite vesicle, the preparation steps of which are largely the same as those in Example 2, except that in step 2, the brain-targeting liposomes obtained in Example 1 and the thylakoid membrane fragments obtained in step 1 are blended at a mass ratio of 1:0.05, and repeatedly extruded through a polycarbonate membrane with a pore size of 100 nm using a liposome extruder to obtain the thylakoid composite vesicle, denoted as "Lip-TK-RVG29@SR9009-3".

[0068] Comparative Example 1. Preparation of blank liposomes

[0069] Soybean lecithin, cholesterol, and DSPE-PEG were dissolved in chloroform at a mass ratio of 7:1:4. The mixture was then placed in a rotary evaporator and evaporated under reduced pressure at 37°C to remove the organic solvent, forming a uniform lipid film on the inner wall of the container. Water was then added, and the mixture was dispersed by ultrasonic oscillation in an ice-water bath to obtain blank liposomes, denoted as Lip.

[0070] Comparative Example 2. Preparation of liposome vesicles containing only the brain-targeting peptide RVG29

[0071] Soybean lecithin, cholesterol, and DSPE-PEG-RVG29 were dissolved in chloroform at a mass ratio of 7:1:4. The mixture was then placed in a rotary evaporator and evaporated under reduced pressure at 37°C to remove the organic solvent, forming a uniform lipid film on the inner wall of the container. Water was then added, and the mixture was dispersed by ultrasonic oscillation in an ice-water bath to obtain brain-targeting liposomes, denoted as "Lip-RVG29".

[0072] Comparative Example 3. Preparation of liposome-based complex vesicles containing only thylakoid membranes

[0073] This comparative example provides a thylakoid composite vesicle, the preparation steps of which are largely the same as those in Example 2, except that in step 2, the brain-targeting liposomes obtained in Example 1 are replaced with blank liposomes prepared in Comparative Example 1, and other conditions remain unchanged. The composite vesicle containing only thylakoid membranes and without RVG29 peptide and SR9009 is obtained by co-extrusion, denoted as "LipTK".

[0074] Comparative Example 4. Preparation of liposome-based complex vesicles without the brain-targeting peptide RVG29

[0075] Soybean lecithin, cholesterol, and SR9009 were dissolved in chloroform at a mass ratio of 7:1:1.2. The mixture was then placed in a rotary evaporator and evaporated under reduced pressure at 37°C to remove the organic solvent, forming a uniform lipid film on the inner wall of the container. Water was then added, and the mixture was dispersed by ultrasonic oscillation in an ice-water bath to obtain liposomes without brain targeting, denoted as "Lip-SR9009".

[0076] A thylakoid composite vesicle was further prepared, and the preparation steps were largely the same as those in Example 2, except that in step 2, the brain-targeting liposomes obtained in Example 1 were replaced with the liposomes Lip-SR9009 prepared in this comparative example, and other conditions remained unchanged. The composite vesicle containing only thylakoid membranes and SR9009 but not RVG29 peptide was obtained by co-extrusion, and was denoted as "LipTK@SR9009".

[0077] Comparative Example 5. Preparation of liposome complex vesicles without REV-ERBα / β agonists

[0078] A thylakoid composite vesicle was prepared, and the preparation steps were largely the same as those in Example 2, except that in step 2, the brain-targeting liposomes obtained in Example 1 were replaced with the liposomes Lip-RVG29 prepared in Comparative Example 2, and other conditions remained unchanged. The composite vesicle containing only thylakoid membranes and RVG29 peptides but not REV-ERBα / β agonists was obtained by co-extrusion, and was denoted as "LipTK-RVG29".

[0079] Experimental Example 1. Particle Size and Potential Detection

[0080] The particle size and potential of four liposome vesicle samples prepared in Examples 1, 2, 3, and 4 were detected using a Malvern potentiometer. The results are as follows: Figure 3 The results show that the hydration dynamics of several vesicles have a particle size of approximately 100 nm (see [reference]). Figure 3 (Part A) and the potentials are both negative (see [reference]). Figure 3 (Part B).

[0081] Experimental Example 2. Flow Cytometry Experiment of Cell Endocytosis

[0082] Using Bend.3 cells, a mouse brain microvascular endothelial cell line, as the target cells, the cellular uptake of four types of vesicles prepared in Examples 1, 2, 3, and 4 was investigated by flow cytometry. Brain-targeting liposomes in the four types of vesicles were labeled with Dio fluorescent dyes. The results are as follows: Figure 4 As shown. Figure 4 Part (A) shows the Dio fluorescence detection results, used to characterize the uptake of lipid vesicles by cells; Figure 4 Section (B) shows the fluorescence detection results of thylakoid membrane TK, used to characterize the entry of thylakoid components into cells. Blank cells (i.e.,...) Figure 4 The blank group and the brain-targeting liposome control group without TK prepared in Example 1 (i.e., the blank group) and the control group without TK prepared in Example 1 (i.e., the blank group) Figure 4 In the 1:0 group, TK fluorescence was almost undetectable. As the TK feeding ratio increased, the proportion of TK-fluorescent positive cells gradually increased. The Lip-TK-RVG29@SR9009-2 prepared in Example 3 (i.e., Figure 4The proportion of TK-positive cells in the 1:0.01 group reached 27.8%, while the proportion of Dio fluorescence-positive cells was 64%; the Lip-TK-RVG29@SR9009-1 prepared in Example 2 (i.e. Figure 4 The proportion of Dio fluorescent positive cells in the 1:0.025 group was 61.8%, and the proportion of TK fluorescent positive cells was 84.0%; the Lip-TK-RVG29@SR9009-3 prepared in Example 4 (i.e., the group with the highest TK feeding ratio) had the highest TK feeding ratio. Figure 4 In the 1:0.05 group, the proportion of TK-positive cells reached 98.0%, but the proportion of Dio-fluorescent positive cells decreased to 46.9% under this condition. In summary, these results suggest that the brain-targeting liposomes and thylakoid membranes in the composite vesicles prepared in Examples 2-4 of this invention at different feed ratios can be effectively taken up by target cells. However, considering all factors, the endocytosis efficiency of the Dio-labeled targeting liposomes (green fluorescence) and the autofluorescence of the thylakoids (red fluorescence) prepared in Example 2 reached a high level, at 61.8% and 84%, respectively, and therefore can be considered the optimal example.

[0083] Experimental Example 3. Determination of drug loading and encapsulation efficiency of SR9009 by high performance liquid chromatography

[0084] The drug loading (loading rate) and encapsulation efficiency of SR9009 in the four samples prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were characterized by liquid chromatography. The results are as follows: Figure 5 As can be seen, the liposomes of Example 1 (corresponding to) Figure 5 Lip-RVG29@SR9009 in Example 1 and the thylakoid composite vesicles of Example 2 (corresponding to) Figure 5 The retention time of the characteristic peak corresponding to SR9009 in Lip TK-RVG29@SR9009 was 5.173 min; the blank lipid vesicles in Comparative Example 1 (corresponding to Figure 5 Lip in Comparative Example 2 and vesicles without SR9009 loading (corresponding to Lip in Comparative Example 2) Figure 5 The presence of no characteristic peak at this retention time for Lip-RVG29 indicates that the lipid carrier and RVG29 peptide do not interfere with the chromatographic detection of SR9009. The samples from Examples 1 and 2 both showed a characteristic absorption peak for SR9009 at 5.173 min, proving that SR9009 was successfully encapsulated within the lipid vesicles. Further based on... Figure 5 Based on the chromatographic peak area and the calculation using the SR9009 standard curve, the SR9009 loading rate of the thylakoid composite vesicles prepared in Example 2 was 1.94%, and the encapsulation rate was 19.4%.

[0085] Experimental Example 4. Verification Experiment of the Synthesis of the Thylakoid Complex Vesicle of the Invention

[0086] The protein components of the four types of vesicles from Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3, as well as the thylakoid membrane precipitate extracted from Example 2, were characterized by SDS-PAGE gel electrophoresis and Western blot. The results are shown in the figure. Figure 6 . Figure 6 The gel electrophoresis results in section (A) show that the thylakoid composite vesicles prepared in Example 2 at a feed ratio of 1:0.025 contain thylakoid membranes (i.e., Figure 6 All feature bands in 5). Figure 6 Part (B) of the Western blot characterization results showed that the blank lipids in Comparative Example 1 (i.e., Figure 6 Liposomes of Comparative Example 1) and Comparative Example 2 (i.e. Figure 6 2) does not contain the thylakoid characteristic proteins H-ATPase and QPRT, thus excluding interference from lipid carriers and targeting peptides; while the liposomes in Comparative Example 3 (i.e. Figure 6 3) and the composite vesicles prepared in Example 2 (corresponding to Figure 6 In all of them, TK (4) appears. Figure 6 Clear bands of the two marker proteins (34 kDa QPRT and 100 kDa H-ATPase) in section 5) demonstrate that the thylakoid membrane proteins of Example 2 and Comparative Example 3 were successfully assembled onto lipid vesicles. Furthermore, even after loading the SR9009 drug and modifying the RVG29 targeting peptide, the thylakoid membrane marker proteins of the composite vesicles in Example 2 remained within the composite vesicles. These experiments demonstrate that Example 2 successfully synthesized a brain-targeting composite vesicle containing SR9009 and thylakoids.

[0087] Experimental Example 5. Treatment of Ischemic Stroke with Thylakoid Complex Vesicles in Mice

[0088] A mouse model was constructed based on the principle of photothrombosis inducing local cerebral thrombosis.

[0089] C57 mice were anesthetized with 2% isoflurane and placed in a stereotactic frame using a gas anesthesia mask, maintained with 1% isoflurane in an oxygen / air mixture. 1% (w / w) rose bengal was administered intravenously 5 minutes before irradiation at a dose of 30 mg / kg. The mice's scalp was shaved, the scalp was cut, and the periosteum was removed. The infarct lesion was formed by irradiation of the right sensorimotor cortex. For illumination, a 532 nm green laser at 25 mW was applied for 10 minutes. At the end of the experiment, the scalp was sutured. The mice were placed in a 37°C constant-temperature blanket until they awoke.

[0090] 1. Fluorescence imaging

[0091] All liposomes used in the fluorescence imaging experiments were modified with DiR for animal imaging. After animal modeling, they were divided into three groups: LipTK@SR9009, LipTK-RVG29@SR9009, and LipTK-RVG29@SR9009+free RVG29 blocking group. The third group was first injected with free RVG29 peptides via tail vein 30 min in advance to block the target binding site, and then injected with the corresponding composite vesicles in the same way as the other groups (the LipTK@SR9009 group was injected with the composite vesicles prepared in Comparative Example 4, and the LipTK-RVG29@SR9009 group and the LipTK-RVG29@SR9009+free RVG29 blocking group were all injected with the composite vesicles prepared in Example 2, and the dosage was 200 μL injected once at a concentration of 4 mg / mL). In vivo fluorescence imaging of the brain was captured at 0 h, 2 h, 4 h, 8 h and 24 h after injection. After 24 h, the animal was dissected, brain tissue was collected, and in vitro fluorescence images were captured.

[0092] The results of detecting DiR fluorescently labeled targeted liposomes in thylakoid complex vesicles are shown in the figure. Figure 7 The results showed that within 24 hours of tail vein injection, the thylakoid complex vesicles prepared in Example 2 at a feed ratio of 1:0.025 effectively penetrated the blood-brain barrier over time under the action of the RVG29 targeting peptide on the surface. When the target site was blocked in advance with free RVG29, the thylakoid complex vesicles were effectively inhibited from entering the mouse brain tissue.

[0093] 2. Treatment efficacy experiment

[0094] Experimental Groups:

[0095] 1-Health Group;

[0096] 2-PBS group;

[0097] 3-LipTK-RVG29 group;

[0098] 4-Lip-RVG29@SR9009 group;

[0099] 5-LipTK-RVG29@SR9009 group;

[0100] 6-Edaravone group.

[0101] Except for the healthy control group, which consisted of healthy mice, all other groups of mice were model mice obtained using the above method.

[0102] Administration and dosage:

[0103] The PBS group was injected with PBS solution;

[0104] The LipTK-RVG29 group was injected with the LipTK-RVG29 composite vesicle solution prepared in Comparative Example 5;

[0105] Lip-RVG29@SR9009 group was injected with the Lip-RVG29@SR9009 liposome solution prepared in Example 1;

[0106] LipTK-RVG29@SR9009 group was injected with the Lip-TK-RVG29@SR9009-1 composite vesicle solution prepared in Example 2;

[0107] The Edaravone group received edaravone injection at a dose of 1 g / kg body weight.

[0108] The concentration of both liposome solution and complex vesicle solution was 4 mg / mL, and the dose was 200 μL each time. All groups were given the drug once every two days for a total of 3 times.

[0109] Five days after tail vein injection treatment, cerebral infarction sites and meningeal tissues from mice in groups 1-5 were collected, ground, and erythrocytes were lysed to extract single cells. These cells were prepared into a single-cell suspension and incubated in DMEM medium. The suspension was then evenly dispersed, and PMA-ionomycin stimulation inhibitor was added to the cell culture medium 3 hours in advance. After antibody staining and fixation every 3 hours for 24 consecutive hours, flow cytometry was performed to analyze the rhythmic patterns of inflammatory factor secretion. Results are shown below. Figure 8 The results showed that in healthy control mice, IL-17 secretion exhibited a typical diurnal sinusoidal fluctuation pattern, with a peak during the day and a trough at night (see...). Figure 8 (The red curve in section (A)). However, 5 days after the onset of the disease, the PBS treatment group showed decreased rhythmic amplitude of IL-17 secretion, increased median levels, and an abnormally elevated nocturnal secretion peak (see [reference]). Figure 8 (Blue curve in section (A)). After treatment with the thylakoid composite vesicle Lip-TK-RVG29@SR9009-1 prepared in Example 2 in the LipTK-RVG29@SR9009 group, the median secretion of IL-17 decreased, tending towards the secretion rhythm of diurnal peak and nocturnal trough in healthy mice (see...). Figure 8 (Part B).

[0110] Seven days after tail vein injection treatment, whole brain tissue was collected from mice in groups 1-5 above, cooled at -20℃ for 5 minutes, and then sectioned. The sections were subsequently stained with 2% TTC at 37℃ for 20 minutes. The staining results are shown below. Figure 9In the image, the infarcted area is unstained and appears white, while normal tissue is stained red. This result shows that, compared with the PBS group, LipTK-RVG29 group, and Lip-RVG29@SR9009 group, the LipTK-RVG29@SR9009 group using the Lip-TK-RVG29@SR9009-1 composite vesicles prepared in Example 2 can effectively reduce the infarct area. Further, whole brain tissue from mice in groups 2-6 above was taken and NAD+ was added. + The extract was homogenized and ground, and the protein was quantified using NAD. + Extraction kit for NAD detection + Level, results see Figure 10 The results showed that, compared with the PBS group, the LipTK-RVG29 group, and the Lip-RVG29@SR9009 group, the LipTK-RVG29@SR9009 group treated with the Lip-TK-RVG29@SR9009-1 vesicle complex vesicles prepared in Example 2 resulted in lower NAD levels after treatment. + The level was significantly elevated, exceeding that of edaravone injection, a commonly used clinical medication.

[0111] 3. Biocompatibility testing:

[0112] Grouping and administration:

[0113] 1- Healthy mice (no drug administered);

[0114] 2- Healthy mice (dosing Lip-TK-RVG29@SR9009-1 composite vesicles prepared in Example 2);

[0115] 3-Model mice (Lip-TK-RVG29@SR9009-1 composite vesicles prepared in Example 2).

[0116] Administer the medication according to the above treatment dosage, one cycle is 5 days, once every 2 days. After 3 administrations, collect whole blood and serum from mice to test blood routine and liver and kidney function. Results are shown below. Figure 11 The results show that the thylakoid composite vesicles prepared in Example 2 have no hepatotoxicity or nephrotoxicity and have good biosafety.

Claims

1. A vesicle-like complex vesicle, characterized in that: It is composed of a mixture of targeted liposomes and thylakoid membrane fragments in a mass ratio of 100:1-5, with a particle size of 80-150 nm; the mass of the thylakoid membrane fragments is based on the mass of chlorophyll they contain; the targeted liposomes are composed of a mixture of neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in a mass ratio of (7-12):1:(0.2-4):(0.82-1.7); the neutral lipids are any one or a mixture of two of phosphatidylethanolamine or phosphatidylcholine; the steroidal lipids are any one or a mixture of two of β-sitosterol or cholesterol; the REV-ERBα / β agonist is any one of SR9011 or SR9009; the brain-targeting modifier containing RVG29 peptide is a conjugate containing RVG29 peptide and membrane-anchored lipid groups.

2. The thylakoid complex vesicle as described in claim 1, characterized in that: The neutral lipid is soybean lecithin; the steroidal lipid is cholesterol.

3. The thylakoid complex vesicle as described in claim 1, characterized in that: The brain-targeting modifier containing RVG29 peptide is a conjugate formed by linking RVG29 peptide and membrane-anchored lipid groups via a hydrophilic linker arm.

4. The thylakoid complex vesicle as described in claim 1, characterized in that: The mass ratio of the neutral lipids, steroidal lipids, brain-targeting modifiers containing RVG29 peptide, and REV-ERBα / β agonists is 7:1:4:1.

2.

5. The thylakoid complex vesicle as described in claim 1, characterized in that: The thylakoid membrane fragments mentioned are derived from any one of spinach, lettuce, or cabbage.

6. The thylakoid complex vesicle as described in claim 1, characterized in that: The mass ratio of the targeted liposomes to the thylakoid membrane fragments is 40:1-1.

5.

7. A method for preparing the thylakoid complex vesicles of claim 1, comprising: 1) Extract thylakoid membrane fragments from the leaves of plants rich in QPRT; 2) Targeted liposomes are prepared by dissolving neutral lipids, steroidal lipids, a brain-targeting modifier containing RVG29 peptide, and a REV-ERBα / β agonist in an organic solvent at a mass ratio of (7-10):1:(1-4):(0.9-1.4); wherein the neutral lipids are any one or a mixture of two of phosphatidylethanolamine or phosphatidylcholine; the steroidal lipids are any one or a mixture of two of β-sitosterol or cholesterol; the REV-ERBα / β agonist is any one of SR9011 or SR9009; and the brain-targeting modifier containing RVG29 peptide is a conjugate containing RVG29 peptide and membrane-anchored lipid groups. 3) The thylakoid membrane fragments obtained in 1) and the targeted liposomes obtained in 2) were blended and extruded at a mass ratio of 1-5:100 to obtain thylakoid composite vesicles with a particle size of 80-150 nm; wherein the amount of thylakoid membrane fragments fed was quantified based on chlorophyll content.

8. The method as described in claim 7, characterized in that: 1) The QPRT-rich plants mentioned above are selected from any one or a mixture of two or more of spinach, lettuce or cabbage.

9. The method as described in claim 7, characterized in that: 3) The mass ratio of the targeted liposomes and thylakoid membrane fragments is 40:1-1.

5.

10. The thylakoid complex vesicles according to any one of claims 1-6, or the thylakoid complex vesicles prepared by the method according to any one of claims 7-9, are used in the preparation of a therapeutic drug for the subacute phase of ischemic stroke; the therapeutic drug is delivered to the ischemic brain microenvironment via intravenous injection to synergistically regulate the multidimensional rhythm parameters of IL-17 secretion by γδT cells.

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