A co-delivery liposome based on whole process regulation and its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, specifically a co-delivery liposome based on full-process regulation and its preparation method and application. The co-delivery liposome of the present invention is composed of an inorganic salt calcium phosphate nano core that can load genes and a liposome phospholipid bilayer with a small molecule drug embedded in the outer layer, and the drug is a histone deacetylase inhibitor propofol-docosahexaenoic acid, which is used for regulating the disordered microglia; the calcium phosphate nano core is loaded with a vascular endothelial growth factor C plasmid for transfection and expression of microglia; the liposome is modified with a selective galactose ligand that activates microglia, which is used for specific targeted activation of galectin-3 receptors on the surface of microglia, and cell selective targeting is achieved; the present invention can effectively regulate the function of inflammatory microglia, promote the generation of blood vessels and meningeal lymphatic vessels, protect cranial nerves and promote regeneration and recovery after TBI, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a liposome for targeting and activating microglia and co-loading drugs, and a preparation method and application thereof. Background Art

[0002] Traumatic brain injury (TBI) is a global public health problem, with more than 50 million patients worldwide each year, placing a huge burden on society and families. Within minutes after TBI, molecular pathways in the damaged area and surrounding normal tissues are activated, leading to neuroinflammation, excitatory amino acid toxicity, mitochondrial dysfunction, oxidative stress, calcium overload, and blood-brain barrier (BBB) ​​disruption, resulting in cerebral ischemia, edema, cytotoxic swelling, and increased intracranial pressure. In the following weeks or even years, the patient's brain is in a state of chronic inflammation for a long time. Studies have shown that the presence of long-term chronic inflammation after TBI is significantly associated with the occurrence of Alzheimer's disease (AD) and cerebrovascular amyloid disease (CAA). Therefore, the long-term presence of chronic inflammation may be one of the main obstacles to regenerative recovery in the later stages of the disease.

[0003] At present, the treatment strategies for TBI mainly focus on anti-inflammatory and antioxidant effects in the acute phase, thereby temporarily alleviating excessive oxidative stress and inflammatory responses in the injured area. However, as the drugs are cleared and metabolized in the body, short-term anti-inflammatory and antioxidant strategies in the acute phase cannot prevent the impact of long-term, complex chronic inflammation on TBI recovery. Therefore, the prognosis is usually poor, and clinical complications occur frequently.

[0004] There is a close functional and structural relationship between cerebral blood vessels and brain nerve cells. Violent brain trauma can lead to cerebral vascular damage or pathological changes, including traumatic cerebral vascular injury and secondary cerebral vascular changes after trauma. Dysfunctional cerebral blood vessels further change the balance of the central nervous system through neurovascular units. Therefore, the remodeling of vascular structure and function in the chronic stage of TBI is also an important aspect of improving the regenerative recovery environment.

[0005] In view of the important role of meningeal lymphatic vessels in promoting the excretion of brain substances discovered in recent years, if the growth of meningeal lymphatic vessels can be promoted and their function of clearing harmful substances from the brain can be enhanced, it is very likely to achieve a long-term regulatory effect on the harmful and repeatedly circulating complex environment during the chronic inflammatory period of TBI, thereby creating a recovery environment that is conducive to the regeneration and survival of neural stem cells, while greatly reducing the risk of complications after TBI.

[0006] There is an inherent unity between the protection and remodeling of blood vessels and the regulation of meningeal lymphatic homeostasis. Blood vessels not only directly exchange substances with the injured area, but the paravascular space formed by astrocytes and the outer wall of blood vessels is also an important part of the "glial lymphatic system". Therefore, the protection and repair of blood vessels can not only restore the material transport and efflux function of the blood vessels themselves, but also further promote the drainage function of meningeal lymphatic vessels, thereby synergistically enhancing the ability of the glial lymphatic pathway to regulate the homeostasis of the brain injury environment.

[0007] The concept of "vascular neural unit" emphasizes the close connection between cerebral blood vessels and central nervous cells, while the concept of "glial lymphatic system" highlights the regulatory effect of meningeal lymphatic drainage on the neural tissue environment. Based on the interconnection between blood vessels and lymphatic vessels, the present invention proposes a "whole vascular system" reconstruction strategy, which aims to autonomously regulate the long-term chronic and complex central environment after TBI in the long term, reduce the risk of complications, and play a role in neuroprotection and promoting neuroregeneration.

[0008] Based on this strategy, the present invention designs a novel liposome that can target and activate microglia and co-load a novel histone deacetylase (HDAC) inhibitor, propofol-docosahexaenoic acid (pro-DHA), and a gene drug, vascular endothelial growth factor C plasmid (pVEGFC). On the one hand, the small molecule drug pro-DHA has a regulatory effect on abnormal microglia in the acute phase, while accelerating the recovery of damaged blood vessels after TBI. On the other hand, the gene drug pVEGFC promotes the remodeling of meningeal lymphatic vessels and enhances its long-term regulatory effect on the brain injury environment. Therefore, this novel liposome can achieve "full process" regulation of the acute and chronic phases after TBI, not only effectively inhibiting the neurological damage caused by inflammation and oxidative stress in the acute phase, but also making key breakthroughs in improving neuroregeneration in the chronic phase of TBI and avoiding related complications, and has good application prospects.

[0009] The present invention uses a calcium phosphate core to load pVEGFC, and then uses dimyristoylphosphatidylcholine (DMPC) liposomes modified with a galactose ligand (Gal) on the surface to co-load the plasmid-containing calcium phosphate core and the small molecule drug pro-DHA to construct a Gal-proDHA / pVEGFC@CaP co-delivery system; this system can have regulatory effects in both the acute and chronic stages of TBI, and effectively achieve nerve protection and functional recovery after TBI. Summary of the invention

[0010] The purpose of the present invention is to provide a co-delivery liposome based on whole-process regulation, which can target and activate microglia and co-load a novel histone deacetylase (HDAC) inhibitor propofol-docosahexaenoic acid (pro-DHA) and a gene drug vascular endothelial growth factor C plasmid (pVEGFC), as well as a preparation method and application thereof.

[0011] The co-delivery liposome based on whole-process regulation provided by the present invention is a nanoscale liposome with a nano-core encapsulated in the inner phase, which is composed of an inorganic calcium phosphate nano-core capable of loading genes and a liposome phospholipid bilayer with a small molecule drug embedded in the outer layer. The drug is a histone deacetylase (HDAC) inhibitor propofol-docosahexaenoic acid (pro-DHA), which can regulate the disordered microglia and promote the recovery of damaged blood vessels; the calcium phosphate (CaP) nano-core encapsulates a vascular endothelial growth factor C plasmid (VEGF-C The co-delivery liposome is composed of a plasmid (pVEGFC), which can achieve transfection and expression of microglia, thereby promoting the remodeling of meningeal lymphatic vessels; the liposome is modified with a selective galactose ligand (galactose, Gal) that activates microglia, which can specifically target the galectin-3 receptor (galectin-3, Gal-3) on the surface of activated microglia, thereby achieving cell-selective targeting; the co-delivery liposome is recorded as Gal-proDHA / pVEGFC@CaP.

[0012] The co-delivered liposomes have full-stage, multi-faceted therapeutic effects on traumatic brain injury, including regulating microglia and promoting vascular and lymphatic remodeling.

[0013] In the present invention, the microglia selective ligand is a galactose ligand, which targets activated microglia by recognizing and activating the galectin-3 receptor (Gal-3) on the surface of microglia. The ligand is connected to the DSPE-PEG2000 amphiphilic fragment and anchored on the liposome by hydrophobic force.

[0014] In the present invention, the galactose ligand material is preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000-galactose (DSPE-PEG2000-Gal).

[0015] In the present invention, the loading of the HDAC inhibitor pro-DHA (a long-chain small molecule therapeutic drug) and the calcium phosphate core encapsulating the pVEGFC plasmid is achieved by dispersing the HDAC inhibitor pro-DHA (a long-chain small molecule therapeutic drug) and the calcium phosphate core encapsulating the pVEGFC plasmid together with the lipid material in a thin film, and then embedding the pro-DHA into the lipid bilayer and encapsulating the pro-DHA into the aqueous phase in the liposome through a hydration process.

[0016] In the present invention, the HDAC inhibitor pro-DHA is synthesized by esterification reaction of propofol and docosahexaenoic acid (DHA). Pro-DHA not only has a considerable microglial inflammation regulating effect, but also has a significant effect of promoting the proliferation and migration of vascular endothelial cells, thereby promoting angiogenesis. The mechanism by which pro-DHA exerts a series of effects may be its excellent HDAC inhibitor activity, so pro-DHA is also a new application of HDAC inhibitors in the field of brain injury.

[0017] In the present invention, the calcium phosphate core containing the plasmid is prepared by a reverse microemulsion method, specifically, an oil-in-water emulsion containing the plasmid and phosphate ions is slowly added to an oil-in-water emulsion containing calcium ions, and after sufficient reaction to form a core, the core is obtained by centrifugation and washing.

[0018] In the present invention, the calcium phosphate-loaded pVEGFC plasmid, whose sequence includes a promoter, a VEGF-C transcription sequence, a prokaryotic resistance gene sequence, and a green fluorescent protein (GFP) sequence, is obtained by amplifying, precipitating, lysing, purifying, and other steps of an engineered E. coli vector. After the pVEGFC plasmid is transfected into microglia, the microglia overexpress and secrete the VEGF-C cytokine, thereby acting on the vascular endothelial growth factor receptor 3 (VEGFR3) on the lymphatic endothelial progenitor cells to promote the formation of meningeal lymphatic vessels.

[0019] In the lipid of the present invention, the calcium phosphate liposome material is selected from one of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, soybean phosphatidylcholine, egg yolk phosphatidylcholine, stearoyloleoylphosphatidylcholine, stearoyllinoleoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, palmitoyllinoleoylphosphatidylcholine, didecanoylphosphatidylcholine, dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine and (2,3-dioleoyl-propyl)-trimethylamine.

[0020] In the present invention, the outer layer liposome material is preferably dimyristoylphosphatidylcholine (DMPC).

[0021] In the present invention, the gene-carrying core used is calcium phosphate nanoparticles, which are encapsulated into liposomes using the reverse microemulsion method and the thin film hydration method. The gene-carrying calcium phosphate core is formed by slowly adding an oil-in-water emulsion containing plasmids and phosphate ions to an oil-in-water emulsion containing calcium ions for full reaction, and the core is mixed with a lipid material and encapsulated in the inner cavity of the liposome by the classic thin film dispersion-hydration method.

[0022] In the present invention, the loaded gene drug is a VEGF-C plasmid, which is loaded by calcium phosphate and then integrated into a novel liposome. After entering the cell, the plasmid enters the cytoplasm through the lysosomal escape effect and then enters the cell nucleus to achieve subsequent transcription and translation, thereby increasing the secretion of the cell VEGF-C cytokine.

[0023] The preparation method of the liposome provided by the present invention comprises the following specific steps:

[0024] (1) Preparing a calcium phosphate nanocore loaded with a gene drug plasmid by a reverse microemulsion method; specifically, adding an oil-in-water emulsion containing a plasmid and phosphate ions to an oil-in-water emulsion containing calcium ions, and after sufficient reaction to form a core, centrifugation and washing are performed;

[0025] (2) The calcium phosphate nanocore is mixed with a lipid material, and calcium phosphate liposomes are prepared by a thin film dispersion method; specifically, the HDAC inhibitor pro-DHA and the calcium phosphate core loaded with pVEGFC plasmid are dispersed together with the lipid material through a thin film, and then embedded into the lipid bilayer and loaded into the aqueous phase of the liposome through a hydration process.

[0026] The specific process of step (1) is as follows:

[0027] Add 4-8 mL CO-520 to 12-16 mL cyclohexane and stir for 15-30 minutes to obtain an oil phase. Add 200-400 μL 2.5 M CaCl 2 The solution was added dropwise to the oil phase to obtain the calcium phase; 50-100 μg of gene drug plasmid was added to 200-400 μL of 12.5 mM Na 2 HPO 4The solution was dispersed in another oil phase, stirred for 10 to 20 minutes, and then 100 to 200 μL of 20 mM DOPA was added, and stirring was continued for 20 to 30 minutes to obtain a phosphate phase; the calcium phase was added to the phosphate phase, and 50 to 100 μL of DOPA was added after 5 to 10 minutes to react for 45 to 60 minutes, followed by adding 30 to 50 mL of anhydrous ethanol to break the emulsion for 20 to 40 minutes, and centrifuged at 12500 to 14500 g for 20 to 40 minutes; after discarding the supernatant, anhydrous ethanol was added for washing, and centrifuged again at 12500 to 14500 g for 20 to 40 minutes; after washing 2 to 3 times, the supernatant was discarded and the precipitate was the calcium phosphate core encapsulating the plasmid; after air drying the precipitate, 1 to 2 mL of chloroform was added to dissolve it and stored at -20°C for later use.

[0028] The specific process of step (2) is as follows:

[0029] Take 500 μL~1 mL CaP chloroform solution, add 1~2 mg DMPC, appropriate amount of DSPE-PEG2000, DSPE-PEG2000-Gal and 4~8 mL chloroform, mix well and vacuum dry at room temperature for 1~1.5 hours, then add 2~4 mL PBS or 5% glucose solution and perform ultrasonic hydration to obtain liposomes, then use an ultrasonic probe to ultrasonicate in an ice bath at a power of 150~250 W for 60~120 s to obtain the target liposomes, which are recorded as Gal-proDHA / pVEGFC@CaP.

[0030] The preparation methods of the other control preparations involved are similar except for the different materials used.

[0031] The in vitro cultured microglial cell line BV2 used in the present invention is a mouse microglioma cell. The cell line retains multiple morphological and functional characteristics of microglial cells and is a commonly used microglial cell line in the field of brain disease research.

[0032] The in vitro cultured neuron cell line HT22 used in the present invention is a mouse hippocampal neuron cell line derived from HT4. This cell line has good applications in in vitro studies of cranial nerve damage and various neurodegenerative diseases.

[0033] The in vitro cultured brain vascular endothelial cell line bEnd.3 used in the present invention is a mouse brain microvascular endothelial cell line, which is commonly used for in vitro blood-brain barrier simulation and angiogenesis ability evaluation of various brain diseases.

[0034] The "traumatic brain injury model" at the cellular level used in the present invention consists of a variety of experimental models such as oxygen-glucose deprivation / reoxygenation (OGD / R), lipopolysaccharide (LPS) induction, Transwell model and scratch experiment.

[0035] The animals used in the present invention are C57 male mice with an age of 6 to 8 weeks, which are common mice species used for controlled cortical impact injury (CCI) models in traumatic brain injury experiments.

[0036] The “traumatic brain injury model” at the animal level used in the present invention adopts the classic controlled cortical impact injury (CCI) model to simulate the disease.

[0037] The present invention describes the preparation process and related characterization of the "whole process" regulated co-delivery liposome Gal-proDHA / pVEGFC@CaP, and provides the evaluation results of its microglial function regulation and vascular and lymphatic vessel remodeling effects at the cellular and animal levels.

[0038] The present invention proves that the liposome has good cell targeting at the cellular level through flow cytometry analysis technology and immunofluorescence imaging technology, and the small molecule pro-DHA can effectively regulate the function of microglia and promote gene transfection of cells. The neuroprotective effect and the ability to promote endothelial cell survival of the liposome are confirmed by MTT and other experiments, and its cytotoxicity is negligible. The preparation has excellent vascular protection and remodeling effects through scratch healing experiments and Transwell resistance measurement experiments.

[0039] The present invention has confirmed in the CCI mouse model that Gal-proDHA / pVEGFC@CaP can effectively accumulate in the brain injury area, and can effectively achieve transfection of cells in the injured area, promote the formation of meningeal lymphatic vessels, and enhance its brain environment regulation. At the same time, Gal-proDHA / pVEGFC@CaP also has good in vivo safety. This "whole process" regulated co-delivery liposome provides a very promising treatment strategy for traumatic brain injury and even a variety of brain injury diseases.

[0040] The co-delivery liposome Gal-proDHA / pVEGFC@CaP constructed by the present invention can utilize its nanosize to cross damaged cerebral blood vessels and infiltrate into the brain, be enriched in the damaged area, and selectively enrich in activated microglia through galactose ligands to reshape the state of microglia and overexpress VEGF-C cytokines, thereby promoting the regeneration of meningeal lymphatic vessels. In addition, a small amount of pro-DHA in the liposomes taken up by vascular endothelial cells can promote the remodeling of damaged blood vessels, thereby achieving the therapeutic goal of "whole vascular" reconstruction together with the remodeling of meningeal lymphatic vessels. The design of the liposome embodies the "full process" intervention concept of brain injury environment regulation that takes into account both the acute and chronic stages of TBI, and organically unifies the theories of inflammatory cell regulation, vascular protection, and lymphatic vessel remodeling, which can effectively improve the therapeutic effect of TBI.

[0041] The novel liposome provided by the present invention co-loads the novel HDAC inhibitor pro-DHA and the gene drug pVEGFC plasmid, which can regulate the function of microglia, protect blood vessels and enhance the harmful substance excretion function of meningeal lymphatic vessels, thereby achieving the effects of neuroprotection, promoting regeneration and reducing complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1-Figure 6 :Characterization of Gal-proDHA / pVEGFC@CaP. Among them:

[0043] Figure 1 This is the structural model diagram of Gal-proDHA / pVEGFC@CaP.

[0044] Figure 2 This is the transmission electron microscopy image of Gal-proDHA / pVEGFC@CaP prepared by the first method.

[0045] Figure 3 Particle size, PDI and Zeta potential of Gal-proDHA / pVEGFC@CaP prepared by the first method.

[0046] Figure 4 The stability of Gal-proDHA / pVEGFC@CaP prepared by the first method (n=3).

[0047] Figure 5 The biocompatibility of the carrier prepared by the first method to BV2 (n=3). * P <0.05, ** P <0.01, or*** P <0.001, **** P <0.0001.

[0048] Figure 6 Particle size, PDI and Zeta potential of Gal-proDHA / pVEGFC@CaP prepared by the second method.

[0049] Figure 7-Figure 12 :Pro-DHA regulates microglia. Among them:

[0050] Figure 7 The inhibitory effect of pro-DHA on microglia (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0051] Figure 8The inhibitory effect of vorinostat on microglia inflammation (n=3). * P <0.05, ** P <0.01, or*** P <0.001, **** P <0.0001.

[0052] Fig. 9 Flow cytometry was used to analyze the effect of pro-DHA on the phenotype ratio of microglia.

[0053] Fig.10 Flow cytometry quantification of the reverse polarization effect of pro-DHA on microglia (n=3). (a) is the control group, (b) is the reverse polarization effect of 10μM pro-DHA on microglia, and (c) is the reverse polarization effect of 50μM pro-DHA on microglia. * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0054] Fig.11 Flow cytometry was used to determine whether pro-DHA promoted microglial phagocytosis (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0055] Fig.12 Flow cytometry was used to measure the effect of vorinostat on microglial phagocytosis (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0056] Figure 13-Figure 19 :Cell targeting and inflammation inhibition of Gal-proDHA / pVEGFC@CaP. Among them:

[0057] Fig.13 Fluorescence confocal analysis of the microglial targeting effect of Gal-proDHA / pVEGFC@CaP (scalebar: 50 µm).

[0058] Fig.14 Fluorescence semi-quantification of Gal-proDHA / pVEGFC@CaP targeting microglia (n=3). *P

[0059] <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0060] Fig.15 Flow cytometry quantification of the targeting efficiency of Gal-proDHA / pVEGFC@CaP after 15 min of saturation with 100 mg / mL galactose (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0061] Fig.16 Flow cytometry analysis of the targeting efficiency of Gal-proDHA / pVEGFC@CaP to activated or inactivated BV2. (a) is the targeting efficiency of Gal-proDHA / pVEGFC@CaP to inactivated BV2, and (b) is the targeting efficiency of Gal-proDHA / pVEGFC@CaP to activated BV2.

[0062] Fig.17 The quantitative results of the targeting efficiency of Gal-proDHA / pVEGFC@CaP to activated or inactivated BV2 (n=3). (a) is the quantitative result of the targeting efficiency of Gal-proDHA / pVEGFC@CaP to inactivated BV2, and (b) is the quantitative result of the targeting efficiency of Gal-proDHA / pVEGFC@CaP to activated BV2. * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0063] Fig.18 The inhibitory effect of each preparation group on iNOS activated microglia (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0064] Fig.19 The ROS inhibition effect of each preparation group on activated microglia (n=3). * P <0.05, **P <0.01, or *** P <0.001, **** P <0.0001.

[0065] Figure 20-22 :Cell transfection efficiency of Gal-proDHA / pVEGFC@CaP. Where:

[0066] Fig. 20 Inverted fluorescence was used to evaluate the transfection efficiency of BV2 with Gal-proDHA / pVEGFC@CaP (scale bar: 100µm).

[0067] Fig.21 Flow cytometric analysis of the transfection efficiency of BV2 with Gal-proDHA / pVEGFC@CaP.

[0068] Fig. 22 Quantitative results of the transfection efficiency of Gal-proDHA / pVEGFC@CaP on BV2 (n=3);

[0069] * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0070] Figure 23-Figure 26 :The vascular protective effect of pro-DHA. Among them:

[0071] Fig.23 The scratch experiment was used to evaluate the effects of pro-DHA and its control drug vorinostat on promoting the proliferation and migration of endothelial cells (the white dotted line is the initial scratch boundary of the cells).

[0072] Fig.24 This is the calculation result of cell fusion rate in scratch test (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0073] Fig.25 The pro-DHA promotes the survival of vascular endothelial cells after OGD / R (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0074] Fig.26 Pro-DHA and its control drug vorinostat promoted the expression of tight junction proteins Claudin-5 and ZO-1 in vascular endothelial cells after OGD / R (scale bar: 100 µm).

[0075] Figure 27-Figure 34 :Vascular protective effect of Gal-proDHA / pVEGFC@CaP. Among them:

[0076] Fig. 27 The scratch experiment was used to evaluate the effects of each preparation group on promoting endothelial cell proliferation and migration (Control: without any treatment; BV2-M0: co-incubated with M0 type BV2; BV2-M1: co-incubated with M1 type BV2 pre-stimulated with LPS; Gal-proDHA / pVEGFC@CaP treated BV2-M1: co-incubated with M1 type BV2 treated with the preparation; the white dotted line is the growth boundary of the cells at that time point).

[0077] Fig.28 This is the calculation result of cell fusion rate in scratch test (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0078] Fig.29 The survival-promoting effects of each group of preparations on vascular endothelial cells after OGD / R (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0079] Fig.30 The effects of each group of preparations on the electrical resistance of vascular endothelial cells after OGD / R (n=3).

[0080] Fig.31 The effects of each group of preparations on the expression of tight junction protein Claudin-5 in vascular endothelial cells after OGD / R (scale bar: 100 µm).

[0081] Fig.32 The fluorescence semi-quantitative results of each group of preparations on the expression of tight junction protein Claudin-5 in vascular endothelial cells after OGD / R (n=3). * P <0.05, ** P <0.01, or *** P<0.001, **** P <0.0001.

[0082] Fig.33 The effects of each group of preparations on the expression of tight junction protein ZO-1 in vascular endothelial cells after OGD / R (scalebar: 100 µm).

[0083] Fig.34 The fluorescence semi-quantitative results of each group of preparations on the expression of tight junction protein ZO-1 in vascular endothelial cells after OGD / R (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0084] Figure 35-Figure 38 :In vivo targeting effect of Gal-proDHA / pVEGFC@CaP. Among them:

[0085] Fig.35 In vivo monitoring of free DiR and DiR-labeled Gal-proDHA / pVEGFC@CaP in small animals at different time points.

[0086] Fig.36 The distribution of free DiR and DiR-labeled Gal-proDHA / pVEGFC@CaP in major organs of mice 72 hours after injection of the preparation (n=3).

[0087] Fig.37 Semi-quantification of fluorescence intensity of free DiR and Gal-proDHA / pVEGFC@CaP labeled with DiR in living animals at different time points (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0088] Fig.38 Semi-quantitative analysis of the fluorescence intensity of free DiR and Gal-proDHA / pVEGFC@CaP labeled with DiR in the main organs of mice 72 hours after injection (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0089] Figure 39-Figure 46:In vivo transfection of Gal-proDHA / pVEGFC@CaP and its effect on promoting meningeal lymphangiogenesis.

[0090] Fig.39 GFP fluorescence expression of Gal-proDHA / pVEGFC@CaP at different doses and administration frequencies in the injured area of ​​mouse brain (scale bar: 50 µm).

[0091] Fig.40 Semi-quantitative analysis of the GFP fluorescence expression intensity of Gal-proDHA / pVEGFC@CaP at different doses and administration frequencies in the injured area of ​​mouse brain (n=3). * P <0.05, ** P <0.01, or *** P <0.001, **** P <0.0001.

[0092] Fig.41 To investigate the safety of the preparation during in vivo transfection (n=3).

[0093] Fig.42 The effect of Gal-proDHA / pVEGFC@CaP at different doses and administration frequencies on mouse meningeal lymphangiogenesis.

[0094] Figure 43-Figure 46 Effects of different doses and administration frequencies of Gal-proDHA / pVEGFC@CaP on the fluorescence area, number of rings, number of sprouts and diameter of meningeal lymphatic vessels in mice (n=3). * P <0.05, ** P <0.01, or *** P <0.001,**** P <0.0001. DETAILED DESCRIPTION

[0095] The present invention is further described below through specific embodiments.

[0096] Example 1: First preparation method and characterization of Gal-proDHA / pVEGFC@CaP

[0097] The first preparation method of Gal-proDHA / pVEGFC@CaP and its control preparation: Gal-CaP, Gal-proDHA@CaP, Gal-pVEGFC@CaP, PEG-proDHA / pVEGFC@CaP and Gal-proDHA / pVEGFC@CaP were prepared by reverse microemulsion method and thin film hydration method.

[0098] Add 6 mL of CO-520 to 14 mL of cyclohexane and stir for 15 minutes to obtain an oil phase. Add 300 μL of 2.5 M CaCl 2 The solution was added dropwise to the oil phase to obtain the calcium phase. 100 μg of plasmid was added to 300 μL of 12.5 mM Na 2 HPO 4 The solution was dispersed in another oil phase, stirred for 10 minutes, and then 100 μL of 20 mM DOPA was added and stirred for 20 minutes to obtain the phosphate phase. The calcium phase was added to the phosphate phase, and 50 μL of DOPA was added after 5 minutes to react for 45 minutes, followed by 40 mL of anhydrous ethanol for demulsification for 20 minutes, and centrifuged at 12500 g for 20 minutes. After discarding the supernatant, anhydrous ethanol was added for washing, and centrifuged again at 12500 g for 20 minutes. After washing 3 times, the supernatant was discarded, and the precipitate was the calcium phosphate core encapsulating the plasmid. After air drying the precipitate, 2 mL of chloroform was added to dissolve it and stored at -20 ° C for later use. Take 500 μL of CaP chloroform solution, add 1 mg of DMPC, appropriate amount of DSPE-PEG2000, DSPE-PEG2000-Gal and 4 mL of chloroform, mix well and dry under vacuum at room temperature for 1 hour, then add 2 mL of PBS or 5% glucose solution and perform ultrasonic hydration, and the obtained liposomes are ultrasonicated in an ice bath at a power of 200 W for 90 seconds using an ultrasonic probe to obtain Gal-proDHA / pVEGFC@CaP liposomes. The preparation methods of the other control preparations are similar except that the materials used are different.

[0099] Particle size, potential and morphology characterization: The particle size, Zeta potential and PDI were measured using a Malvern laser particle size analyzer, and the morphology and size were observed under a transmission electron microscope (TEM).

[0100] Preparation stability study: Gal-proDHA / pVEGFC@CaP was dispersed in PBS or PBS containing 10% FBS and allowed to stand at 4°C for 14 days. Appropriate amounts of the preparation to be tested were taken out on days 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 14, and the changes in the particle size of the preparation over time were monitored using a Malvern laser particle size analyzer.

[0101] Investigation of carrier biocompatibility: BV2 cells were seeded in 96-well plates and incubated with different concentrations of formulation carriers for 24 h. The cell survival rate was determined using the classic MTT method.

[0102] The results show that: Figure 2 As shown in the figure, the prepared liposomes are relatively regular spherical in shape, uniform in size, and have a particle size of about 40 nm. Figure 3As shown, the particle size of Gal-proDHA / pVEGFC@CaP measured by Malvern particle size analyzer was about 32.30 nm, PDI was 0.319, the particle size was uniform, and the Zeta potential was about -2.15 mV, close to neutral, indicating that the liposomes were successfully prepared.

[0103] like Figure 4 As shown, the particle size of Gal-proDHA / pVEGFC@CaP remained basically unchanged within two weeks whether in PBS or PBS containing 10% FBS, proving that the liposomes had good stability.

[0104] like Figure 5 As shown, Gal-proDHA / pVEGFC@CaP did not exhibit obvious cytotoxicity to BV2 cells within a concentration of 0.1 mg / mL, demonstrating that the liposomes have good safety and biocompatibility at therapeutic concentrations.

[0105] Example 2: Second preparation method and characterization of Gal-proDHA / pVEGFC@CaP

[0106] Preparation method of Gal-proDHA / pVEGFC@CaP and its control preparation: Gal-CaP, Gal-proDHA@CaP, Gal-pVEGFC@CaP, PEG-proDHA / pVEGFC@CaP and Gal-proDHA / pVEGFC@CaP were prepared by reverse microemulsion method and thin film hydration method.

[0107] Add 6 mL of CO-520 to 14 mL of cyclohexane and stir for 15 minutes to obtain an oil phase. Add 300 μL of 2.5 M CaCl 2 The solution was added dropwise to the oil phase to obtain the calcium phase. 80 μg of plasmid was added to 300 μL of 12.5 mM Na 2 HPO 4The solution was dispersed in another oil phase, stirred for 15 minutes, and then 200 μL of 20 mM DOPA was added and stirred for 25 minutes to obtain the phosphate phase. The calcium phase was added to the phosphate phase, and 100 μL of DOPA was added after 5 minutes to react for 45 minutes, followed by 40 mL of anhydrous ethanol for demulsification for 30 minutes, and centrifuged at 13500 g for 30 minutes. After discarding the supernatant, anhydrous ethanol was added for washing, and centrifuged again at 13500 g for 30 minutes. After washing twice, the supernatant was discarded, and the precipitate was the calcium phosphate core encapsulating the plasmid. After air drying the precipitate, 2 mL of chloroform was added to dissolve it and stored at -20 ° C for later use. Take 1 mL of CaP chloroform solution, add 2 mg of DMPC, appropriate amount of DSPE-PEG2000, DSPE-PEG2000-Gal and 8 mL of chloroform, mix well and dry under vacuum at room temperature for 1.5 hours, then add 2 mL of PBS or 5% glucose solution and perform ultrasonic hydration. The obtained liposomes are ultrasonicated in an ice bath at a power of 220 W for 75 s using an ultrasonic probe to obtain Gal-proDHA / pVEGFC@CaP liposomes. The preparation methods of the other control preparations are similar except that the materials used are different.

[0108] Particle size, zeta potential and PDI were determined using a Malvern laser particle size analyzer.

[0109] The results show that: Figure 6 As shown, the particle size of Gal-proDHA / pVEGFC@CaP measured by Malvern particle size analyzer was about 31.63 nm, the PDI was 0.213, the particle size was uniform, and the Zeta potential was about -1.22 mV, close to neutral, indicating that the liposomes were successfully prepared.

[0110] Example 3: Evaluation of the microglial regulatory function of the novel small molecule drug pro-DHA

[0111] Pro-DHA inhibits inflammation of microglia: Since the mechanism of pro-DHA may be derived from its HDAC inhibitor activity, in order to explore its advantages over traditional HDAC inhibitors, the commercially available HDAC inhibitor vorinostat was selected as a free control drug. BV2 cells were pretreated with 100 ng / ml LPS for 24 hours, resuspended with DMEM complete medium containing different concentrations of pro-DHA or vorinostat, inoculated in 24-well plates and continued to be cultured for 24 hours. The untreated group and the LPS-treated group were used as negative and positive controls, respectively. After 24 hours, the cells in each group were collected, centrifuged and washed twice with PBS, and then 100 μL CD80 and CD206 antibodies were added. They were incubated in an ice bath in the dark for 45 minutes, and CD80 and CD206 positive cells were detected by flow cytometry to examine the proportion of M1 and M2 microglia.

[0112] Pro-DHA enhances the phagocytic effect of microglia: PLGA nanoparticles loaded with fluorescent dye coumarin 6 (cou-6) were prepared at a ratio of 10 mg PLGA and 10 μg cou-6. BV2 cells were cultured in a medium containing or without 10 μM pro-DHA or vorinostat for 24 h, and then in a medium containing PLGA nanoparticles loaded with fluorescent dye cou-6, the nanoparticle concentration was 50 ng / mL in terms of cou6, and placed at 37°C and 5% CO 2 The cells were incubated in a cell culture incubator for 30 min, centrifuged and the supernatant was discarded, the cells were washed once with PBS, and resuspended with 200 μL PBS. The fluorescence intensity of cou6 uptake by BV2 cells was detected by flow cytometry.

[0113] The results show that: Figure 7-8 As shown in the figure, both pro-DHA and vorinostat effectively reduced the proportion of CD80 (M1 microglial marker) positive cells at a concentration of 10 μM, indicating that both have significant inhibitory effects on inflammation; Figure 9-10 As shown, after treatment with 10 μM pro-DHA, the M1 / M2 microglia ratio was significantly reduced, indicating that 10 μM pro-DHA can effectively reverse M1 microglia into M2 microglia and improve the inflammatory environment.

[0114] like Figure 11-12 As shown, 10 μM pro-DHA effectively enhanced the fluorescence intensity of microglial cells after taking up cou-6-containing PLGA nanoparticles, while vorinostat had no enhancement at all concentrations, indicating that pro-DHA at therapeutic concentrations showed the potential to enhance the phagocytic function of microglia, which is not possessed by vorinostat.

[0115] Example 4: Microglial selective targeting ability and inflammation inhibition effect of Gal-proDHA / pVEGFC@CaP

[0116] Flow cytometry to investigate the selective targeting ability of Gal-proDHA / pVEGFC@CaP on microglia: BV2 cells were pretreated with 100 ng / ml LPS for 24 h, and incubated with PEG-proDHA / pVEGFC@CaP or Gal-proDHA / pVEGFC@CaP containing 10 μM pro-DHA and an equal amount of DiI fluorescent probe for 1 h or 2 h, and the cells were collected, washed twice with PBS after centrifugation, and the uptake of different preparations by BV2 cells was detected by flow cytometry. To investigate the targeting mechanism, cells under the same conditions were first incubated with a medium containing 100 μg / mL galactose for 15 min to saturate the Gal receptors on the cell surface, and then the preparations were added and incubated for 1 h and 2 h, and the cells were collected, washed twice by centrifugation, and the uptake of different preparations by BV2 cells was detected by flow cytometry.

[0117] Fluorescence confocal imaging to investigate the microglial selective targeting of Gal-proDHA / pVEGFC@CaP: BV2 cells were pre-seeded in confocal culture dishes and pre-treated with 100 ng / ml LPS for 24 h.

[0118] Gal-proDHA / pVEGFC@CaP were co-incubated for 1 h, the culture medium was removed, and the cells were washed twice with PBS. Then, 200 μL of PBS was added to keep the cells moist, and the cells were placed under a fluorescence confocal microscope to observe the preparation uptake.

[0119] Inhibitory effect of Gal-proDHA / pVEGFC@CaP on microglial inflammation: BV2 cells were seeded in 96-well plates and pretreated with 100 ng / ml LPS for 24 h. They were then incubated with free small molecule drugs pro-DHA, Gal-CaP, Gal-proDHA@CaP, Gal-pVEGFC@CaP, PEG-proDHA / pVEGFC@CaP and Gal-proDHA / pVEGFC@CaP containing 10 μM pro-DHA equivalents for 24 h. The culture medium was removed and the untreated group and the LPS-treated group were used as negative and positive controls, respectively. The expression levels of inflammatory markers iNOS and ROS in each group were detected using inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) detection kits.

[0120] The results show that: Figure 13-14 As shown, there was no significant difference in the uptake of the two preparations with and without ligand modification by microglia without LPS activation, while the uptake of ligand-modified Gal-proDHA / pVEGFC@CaP by microglia activated by LPS was about 6 times that of unmodified PEG-proDHA / pVEGFC@CaP, proving that the modification of galactose ligand can effectively promote the selective targeting of liposomes to activated microglia. Figure 16-17 The flow cytometric analysis results also showed similar conclusions. Fig.15 As shown, to further support the targeting effect of galactose ligands, after the activated microglia were saturated with galactose on the surface of galectin-3 receptors, the targeting effect of Gal-proDHA / pVEGFC@CaP was masked, further confirming the cell targeting ability provided by galactose ligands.

[0121] Based on the excellent targeting ability of Gal-proDHA / pVEGFC@CaP, the liposome may further enhance the inflammatory regulation ability of the loaded drug pro-DHA. Fig.18 As shown in the figure, the level of iNOS in BV2 cells increased significantly after LPS stimulation. After treatment with each drug group, except for the empty liposome group, which showed no significant improvement, the level of iNOS in the cells decreased to a certain extent, but the decrease in the Gal-proDHA / pVEGFC@CaP treatment group was the most obvious, returning to a level comparable to that of normal cells. Fig.19 As shown in the figure, after BV2 cells were treated with OGD / R, the intracellular ROS level increased significantly, indicating that the OGD / R model was successfully constructed. After being treated with PEG-proDHA / pVEGFC@CaP or Gal-proDHA / pVEGFC@CaP, the level of ROS in cells decreased to a certain extent, but Gal-proDHA / pVEGFC@CaP had a more significant effect on downregulating ROS. The above results all show that Gal-proDHA / pVEGFC@CaP can significantly enhance the therapeutic effect of pro-DHA and has the most effective effect on regulating microglial inflammation.

[0122] Example 5: Evaluation of the microglial transfection effect of Gal-proDHA / pVEGFC@CaP

[0123] Flow cytometric analysis of microglial transfection of Gal-proDHA / pVEGFC@CaP: BV2 cells were seeded in 12-well plates, pretreated with 100 ng / ml LPS for 24 h, and incubated with different preparations for 24 h. The cells were collected, centrifuged and washed twice with PBS. The transfection effects of different preparations on BV2 cells were detected by flow cytometry.

[0124] Inverted fluorescence analysis of microglial cell transfection of Gal-proDHA / pVEGFC@CaP: BV2 cells were seeded in 6-well plates, pretreated with 100 ng / ml LPS for 24 h, and incubated with liposome Gal-proDHA / pVEGFC@CaP loaded with different amounts of plasmid for 24 h. The culture medium was aspirated and the cells were washed twice with PBS. 200 μL of PBS was added to each well to keep the cells moistened, and the transfection effect of Gal-proDHA / pVEGFC@CaP with different plasmid loading amounts on BV2 cells was examined under an inverted fluorescence microscope.

[0125] The results show that: Fig. 20 As shown in the figure, compared with liposomes without ligand modification, Gal-proDHA / pVEGFC@CaP, which can increase cellular uptake, has a higher transfection efficiency for activated BV2 cells and shows a dose-dependent manner of the plasmid. Figure 21-22 As shown, flow cytometry analysis further confirmed the promoting effect of galactose targeting modification on liposome transfection.

[0126] Example 6: Evaluation of the vascular protective effect of Gal-proDHA / pVEGFC@CaP

[0127] Investigation of pro-DHA's vascular protection: Scratch experiment was used to investigate the effects of different concentrations of pro-DHA and vorinostat in promoting the migration and proliferation of vascular endothelial cells; MTT method was used to determine the pro-survival effects of different concentrations of pro-DHA on OGD / R vascular endothelial cells; the expression of tight junction proteins Claudin-5 and ZO-1 in OGD / R vascular endothelial cells after treatment with different concentrations of free pro-DHA or vorinostat was investigated.

[0128] Evaluation of Gal-proDHA / pVEGFC@CaP in promoting the survival of vascular endothelial cells: Mouse vascular endothelial cells bEnd.3 were seeded in a 96-well plate, and the culture medium was aspirated after they were fully attached to the wall. The cells were washed twice with PBS, and cultured in a sugar-free and serum-free culture medium at a constant temperature for 9 hours under a hypoxic environment. Then, they were cultured in a culture medium containing different preparation groups of 0.5 μM pro-DHA under a normal oxygen environment for 24 hours. Cells in a normal aerobic environment were used as positive controls, and OGD / R cells without preparation treatment were used as negative controls. The classic MTT method was used to evaluate cell survival rate.

[0129] Investigation of the protective effect of Gal-proDHA / pVEGFC@CaP on vascular endothelial cells: Mouse vascular endothelial cells bEnd.3 were inoculated in the upper chamber of 0.4μm Transwell and cultured in a 12-well plate. The cell resistance was measured every day using a resistance meter. When the resistance reached 200Ω, the culture medium was aspirated and the cells were washed twice with PBS. After constant temperature culture for 9 hours in a sugar-free and serum-free culture medium under anoxic environment, they were continued to be cultured for 24 hours in a normal oxygen environment in the culture medium of different preparation groups containing 0.5μM proDHA. Cells in normal aerobic environment were used as positive controls, and OGD / R cells without preparation treatment were used as negative controls. A resistance meter was used to monitor the changes in cell resistance of each group over time.

[0130] Investigation of Gal-proDHA / pVEGFC@CaP in promoting endothelial cell migration and proliferation: To investigate the dose-effect relationship of free pro-DHA on endothelial cell migration and proliferation, mouse endothelial cells bEnd.3 were inoculated in 12-well plates, and uniform scratches were made in the wells using a 1 mL pipette tip. After washing twice with PBS, they were incubated with proDHA containing different concentrations, and the changes in the scratches were observed under an inverted fluorescence microscope in phase contrast mode at 12 h, 24 h, and 36 h. To investigate the indirect mechanism of different preparations in promoting endothelial cell migration and proliferation, BV2 cells were inoculated in the upper chamber of 0.4 μm Transwell, pretreated with 100 ng / ml LPS for 24 h, and then incubated with different preparations containing 0.5 μM proDHA, and then cultured in 12-well plates with scratched cells, and the changes in the scratches in the lower chamber were observed under an inverted fluorescence microscope in phase contrast mode at 12 h, 24 h, and 36 h.

[0131] Investigation of Gal-proDHA / pVEGFC@CaP enhancing the tight junction of vascular endothelial cells: Mouse vascular endothelial cells bEnd.3 were seeded in a 12-well plate and cultured in a sugar-free and serum-free medium at a constant temperature under an oxygen-free environment for 9 hours. Then, they were cultured in a medium containing different preparations of 0.5 μM pro-DHA under a normal oxygen environment for 24 hours, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, blocked with anti-fluorescence quenching blocking solution at room temperature for 1 hour, added with a 100-fold diluted ZO-1 or Claudin-5 primary antibody solution, incubated at 4°C overnight, removed the primary antibody, washed three times with PBS, added with a 200-fold diluted fluorescent secondary antibody solution, incubated at room temperature for one hour, washed three times with PBS, and cells in a normal aerobic environment were used as positive controls, and OGD / R cells without preparation treatment were used as negative controls. The expression levels of tight junction proteins ZO-1 or Claudin-5 in each group of cells were examined using an inverted fluorescence microscope.

[0132] The results show that: Figure 23-24 As shown in the figure, 2 μM pro-DHA significantly enhanced the migration and proliferation of endothelial cells, and the cell fusion rates at 24h and 36h reached 77.9% and 88.3%, respectively, while the control drug vorinostat showed no significant effect. Fig.25 As shown in the figure, after treatment with 0.5μM and 2μM pro-DHA, the survival rate of OGD / R cells increased from 43.8% to 71.1% and 82.3%, significantly enhancing the activity of vascular endothelial cells. Fig.26As shown in the figure, after treating OGD / R vascular endothelial cells with pro-DHA at various concentrations, the tight junction proteins Claudin-5 and ZO-1 concentrated on the cell membrane were significantly improved, while vorinostat had no obvious effect. The above results prove that free small molecule pro-DHA has a significant vascular protective effect compared with the control drug.

[0133] The enhancement effect of carrier loading on the effect of pro-DHA was investigated. Figure 27-28 As shown in the figure, Gal-proDHA / pVEGFC@CaP treatment of M1 microglia significantly enhanced the migration and proliferation of vascular endothelial cells, and the cell fusion rates at 24h and 36h reached 41.9% and 81.4%, respectively. Fig.29 As shown in the figure, Gal-proDHA / pVEGFC@CaP increased the survival rate of vascular endothelial cells treated with OGD / R from 45.3% to 84.3%, while the other drug groups had no significant effect. Fig.30 As shown in the data, after OGD / R treatment, the resistivity of bEnd.3 cells decreased to 79.3% within 6 hours after reoxygenation. Among the drug groups, only Gal-proDHA / pVEGFC@CaP could significantly reverse the downward trend of resistivity and promote the increase of resistivity 6 hours after reoxygenation, making the resistivity reach 107.8% at 24 hours, indicating that Gal-proDHA / pVEGFC@CaP can effectively prevent endothelial cell death and promote angiogenesis.

[0134] like Figure 31-34 As shown in the data, after OGD / R treatment, the tight junction proteins Claudin-5 and ZO-1 of vascular endothelial cells were severely lost. After pro-DHA treatment, the fluorescence intensity of Claudin-5 in cells increased by nearly 3 times. The empty group had no obvious effect, while the preparation group increased the fluorescence intensity of Claudin-5 by nearly 6 times, and the fluorescence intensity of ZO-1 by nearly 7 times. This proves that Gal-proDHA / pVEGFC@CaP can effectively promote the expression of tight junction proteins in vascular endothelial cells, maintain vascular tightness, and protect vascular function.

[0135] Example 7: In vivo brain targeting evaluation of Gal-proDHA / pVEGFC@CaP

[0136] Establishment of CCI model in mice: After anesthesia, 6-8-week-old mice were placed in a prone position on a stereotaxic apparatus, and the coronal and sagittal sutures of the parietal bones were fully exposed under sterile conditions. A 3.5 mm diameter bone window (3 mm diameter of the striking cap) was milled 1 mm behind the bregma and 2 mm to the right of the sagittal suture. The parameters were set as a striking speed of 3.5 m / s, a dwell time of 400 ms, and a striking depth of 1.0 mm. In the sham operation group, only a bone window was milled, and the rest of the operation was the same as before. After suturing, the wound was disinfected and the mice were kept until they woke up.

[0137] To investigate whether the liposomes can target the damaged brain area in the CCI model, the experimental mice were randomly divided into two groups (n=3) one day after modeling. The two groups were injected with free DiR fluorescent probe and DiR-labeled liposomes (DiR dosage was 1 mg / kg), respectively. The DiR fluorescence in the mouse brain was detected by in vivo imaging at 1h, 2h, 4h, 8h, 12h, 24h, 48h and 72h to investigate the brain penetration ability of liposomes.

[0138] To investigate the distribution of the liposomes in various organs, mice were routinely treated 72 hours after injection of free DiR fluorescent probe or DiR-labeled liposomes, and the main organs (brain, heart, liver, spleen, lung, and kidney) were removed after cardiac perfusion with normal saline and 4% paraformaldehyde (PFA), and the DiR fluorescence of each organ was detected using an in vivo imaging device.

[0139] The results show that: Fig.35 and Fig.37 As shown in the figure, free DiR did not accumulate significantly in the brain, but Gal-proDHA / pVEGFC@CaP began to accumulate in the injured area of ​​the brain 1 hour after injection, and the accumulation increased significantly over time, reaching a peak at 12 hours, but it could maintain a high accumulation in the brain for at least 72 hours. Fig.36 and Fig.38 As shown in the figure, the preparation is mainly cleared through the liver, and the accumulation in the liver is significantly higher than that in the spleen and kidney. The above data confirm that Gal-proDHA / pVEGFC@CaP has good targeting ability to the brain injury site and can effectively improve the treatment efficiency of brain injury.

[0140] Example 8: In vivo transfection of Gal-proDHA / pVEGFC@CaP and evaluation of promoting meningeal lymphangiogenesis

[0141] In vivo transfection effect of Gal-proDHA / pVEGFC@CaP: After modeling, the experimental mice were randomly divided into 6 groups (n=3) and given different dosing frequencies and doses of PEG-proDHA / pVEGFC@CaP or Gal-proDHA / pVEGFC@CaP. 24 hours after the last administration, the mice were treated routinely, and the brains were taken after cardiac perfusion with normal saline and 4% paraformaldehyde (PFA), immersed in 4% paraformaldehyde overnight, immersed in sucrose solution gradient dehydration, and then embedded in OCT compound and frozen at -20℃, and then sliced ​​using a cryostat. The expression of GFP fluorescent protein in brain slices was observed under an inverted fluorescence microscope.

[0142] Effect of Gal-proDHA / pVEGFC@CaP on promoting meningeal lymphangiogenesis: To investigate whether the in vivo transfection can effectively promote the formation of meningeal lymphangiogenesis, the experimental mice were randomly divided into 6 groups (n=3) after modeling, and PEG-proDHA / pVEGFC@CaP or Gal-proDHA / pVEGFC@CaP with different dosing frequencies and doses were given. After 28 days of administration, the mice were treated routinely, and the skull was removed after cardiac perfusion with normal saline and 4% paraformaldehyde (PFA), and soaked in 2% paraformaldehyde overnight. The dura mater was carefully peeled off in PBS and spread on a glass slide, washed three times with PBS, and 0.25% Triton X-100 (v / v) solution was added to the surface of the meninges for incubation for 10 minutes. After PBS washing, 10% goat serum (v / v) in PBS solution was used to block at room temperature for 2 hours, and then the meninges were incubated with Lyve-1 antibody dilution at 4°C overnight. After washing the meninges with PBS, the sections were incubated with fluorescent secondary antibody dilution for 1 h, washed twice with PBS, an appropriate amount of DIPI-containing sealing solution was added, and a coverslip was gently covered. The formation of meningeal lymphatic vessels was observed using a fluorescent confocal microscope in full scan mode.

[0143] The results show that: Figure 39-40 As shown, plasmid transfection was successfully achieved in each group of mice, the fluorescence intensity of GFP fluorescent protein in the mouse brain was significantly increased, and the expression of GFP fluorescent protein was positively correlated with the dosage and frequency of administration. Fig.41 As shown, there was no significant fluctuation in the body weight of mice at various dosages and frequencies, indicating that the in vivo transfection of Gal-proDHA / pVEGFC@CaP has excellent safety.

[0144] like Figure 42-46As shown, the formation of mouse meningeal lymphangiogenesis was enhanced to a certain extent in each dosing dose and frequency group. Under the dosing regimen of 50 μg plasmid load and 3 times a week, the fluorescence area, number of rings, number of buds and diameter of mouse meningeal lymphatic vessels were significantly improved. Taking into account the actual clinical use, the dosing regimen of 50 μg plasmid load and 3 times a week was finally selected.

[0145] The above data prove that Gal-proDHA / pVEGFC@CaP can achieve in vivo transfection, promote the expression of VEGF-C, effectively enhance the formation of meningeal lymphatic vessels, enhance its function of regulating the brain injury environment, and thus promote the recovery of brain injury.

[0146] Examples 4-8 are the results of the performance test of Gal-proDHA / pVEGFC@CaP prepared in Example 1. There is no significant difference in the performance test results of Gal-proDHA / pVEGFC@CaP prepared in Example 2. The test results show that: the liposome has the effect of inhibiting microglial inflammation, can selectively target activated microglia in vitro, achieve gene transfection of microglia, and play a good vascular protection role; it has the ability to target brain injury sites in vivo, can achieve gene transfection of injury sites, and promote the remodeling of meningeal lymphatic vessels. In addition, the liposome Gal-proDHA / pVEGFC@CaP prepared in Example 2 has good in vitro and in vivo safety.

Claims

1. A co-delivery liposome based on full process regulation, characterized in that: The invention relates to a nanoscale liposome with a nano-core encapsulated in the inner phase, which is composed of an inorganic calcium phosphate nano-core capable of loading genes and a liposome phospholipid bilayer with a small molecule drug embedded in the outer layer. The drug is a histone deacetylase (HDAC) inhibitor propofol-docosahexaenoic acid (pro-DHA), which is used to regulate dysregulated microglia and promote the recovery of damaged blood vessels; the calcium phosphate (CaP) nano-core is loaded with a vascular endothelial growth factor C plasmid (pVEGFC), which is used to transfect and express microglia and promote the remodeling of meningeal lymphatic vessels; the liposome is modified with a selective galactose ligand (Gal) for activating microglia, which is used to specifically target and activate the galectin-3 receptor (Gal-3) on the surface of microglia to achieve cell-selective targeting; the co-delivery liposome is referred to as Gal-proDHA / pVEGFC@CaP.

2. The co-delivery liposome based on whole process regulation according to claim 1, characterized in that: The microglia selective ligand is a galactose ligand, which targets activated microglia by recognizing and activating the galectin-3 receptor (Gal-3) on the surface of microglia; the ligand is connected to the DSPE-PEG2000 amphiphilic fragment and anchored on the liposome by hydrophobic force.

3. The co-delivery liposome based on whole process regulation according to claim 1, characterized in that: The loading of the histone deacetylase inhibitor propofol-docosahexaenoic acid and the calcium phosphate core containing the vascular endothelial growth factor C plasmid is achieved by dispersing the film together with the lipid material, and then embedding into the lipid bilayer and encapsulating into the aqueous phase of the liposome through the hydration process.

4. The co-delivery liposome based on whole process regulation according to claim 1, characterized in that: The histone deacetylase inhibitor propofol-docosahexaenoic acid is synthesized by esterification reaction of propofol and docosahexaenoic acid (DHA).

5. The co-delivery liposome based on whole process regulation according to claim 1, characterized in that: The calcium phosphate core loaded with vascular endothelial growth factor C plasmid is prepared by a reverse microemulsion method, specifically, an oil-in-water emulsion containing plasmid and phosphate ions is added to an oil-in-water emulsion containing calcium ions, and after sufficient reaction to form a core, the core is obtained by centrifugation and washing.

6. The co-delivery liposome based on whole process regulation according to claim 1, characterized in that: The calcium phosphate nano core package carries a vascular endothelial growth factor C plasmid, the sequence of which includes a promoter, a VEGF-C transcription sequence, a prokaryotic resistance gene sequence and a green fluorescent protein (GFP) sequence, and is obtained by amplifying, precipitating, lysing and purifying an engineered Escherichia coli vector.

7. A method for preparing co-delivery liposomes based on full process regulation as claimed in any one of claims 1 to 6, characterized in that: The specific steps are: (1) preparing a calcium phosphate nanocore loaded with a gene drug plasmid by a reverse microemulsion method; specifically, adding an oil-in-water emulsion containing a plasmid and phosphate ions to an oil-in-water emulsion containing calcium ions, and after sufficient reaction to form a core, centrifugation and washing are performed; (2) The calcium phosphate nanocore is mixed with a lipid material, and calcium phosphate liposomes are prepared by a thin film dispersion method; specifically, the HDAC inhibitor pro-DHA and the calcium phosphate core loaded with pVEGFC plasmid are dispersed together with the lipid material by thin film dispersion, and then embedded into the lipid bilayer and loaded into the aqueous phase of the liposome by a hydration process.

8. The preparation method according to claim 7, characterized in that: The specific process of step (1) is as follows: Add 4-8 mL CO-520 to 12-16 mL cyclohexane and stir for 15-30 minutes to obtain an oil phase; add 200-400 μL 2.5 M CaCl2 solution dropwise to the oil phase to obtain a calcium phase; add 50-100 μg of gene drug plasmid to 200-400 μL 12.5 mM Na2HPO4 solution, disperse in another oil phase, stir for 10-20 minutes, add 100-200 μL 20 mM DOPA, continue stirring for 20-30 minutes to obtain a phosphate phase; add the calcium phase to the phosphate phase, and after 5-10 minutes, add 50-100 μL DOPA was reacted for 45 to 60 minutes, and then 30 to 50 mL of anhydrous ethanol was added to break the emulsion for 20 to 40 minutes, and centrifuged at 12500 to 14500 g for 20 to 40 minutes; after discarding the supernatant, anhydrous ethanol was added for washing, and centrifuged again at 12500 to 14500 g for 20 to 40 minutes; after washing 2 to 3 times, the supernatant was discarded and the precipitate was the calcium phosphate core encapsulating the plasmid; after air drying the precipitate, 1 to 2 mL of chloroform was added to dissolve it, and it was stored at -20°C for later use; The specific process of step (2) is as follows: Take 500 μL~1 mL CaP chloroform solution, add 1~2 mg DMPC, appropriate amount of DSPE-PEG2000, DSPE-PEG2000-Gal and 4~8 mL chloroform, mix well and dry under vacuum at room temperature for 1~1.5 hours, then add 2~4 mL PBS or 5% glucose solution and perform ultrasonic hydration to obtain liposomes, and then use an ultrasonic probe to ultrasonicate in an ice bath at a power of 150~250 W for 60~120 s to obtain the target liposomes, which are recorded as Gal-proDHA / pVEGFC@CaP.

9. Use of the co-delivery liposome based on global regulation as claimed in any one of claims 1 to 6 in the preparation of a gene drug delivery system for treating traumatic brain injury.

Images