WO2021098606A1 - 靶向颅脑损伤病灶的脂质纳米递药系统及其制备方法和应用 - Google Patents

靶向颅脑损伤病灶的脂质纳米递药系统及其制备方法和应用 Download PDF

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WO2021098606A1
WO2021098606A1 PCT/CN2020/128748 CN2020128748W WO2021098606A1 WO 2021098606 A1 WO2021098606 A1 WO 2021098606A1 CN 2020128748 W CN2020128748 W CN 2020128748W WO 2021098606 A1 WO2021098606 A1 WO 2021098606A1
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delivery system
drug delivery
cyclosporin
lipid
lipid nano
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French (fr)
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高小玲
宋清香
江淦
陈乐培
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上海交通大学医学院
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Priority to EP20890093.6A priority patent/EP4062940A4/en
Priority to US17/776,217 priority patent/US20220401362A1/en
Publication of WO2021098606A1 publication Critical patent/WO2021098606A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to the field of technical nano-biomedical material technology-nano medicine and drug delivery system, in particular to a lipid nano-drug delivery system targeting brain injury lesions, and a preparation method and application thereof.
  • Traumatic brain injury is a common disease in neurosurgery, and its high mortality and disability rate bring great economic and psychological burdens to patients' families.
  • TBI damage can be divided into two types: primary and secondary.
  • Primary damage mainly occurs at the point of action of external force, and brain parenchymal damage caused by mechanical damage.
  • the damage to the brain not only occurs at the moment of injury.
  • Early mechanical damage often induces secondary injury cascades, including cell excitotoxicity, vascular and cytotoxic edema, hypoxia-ischemia, mitochondrial dysfunction, and oxidative stress. Excitation and inflammation exacerbate the damage of the original damaged tissue.
  • Due to the lack of effective treatment methods many patients with craniocerebral injury are left with neurological dysfunction of varying degrees after their consciousness is restored. However, there is no specific treatment plan to improve the neurological dysfunction caused by traumatic brain injury.
  • the pathophysiological mechanism of TBI neurological dysfunction is multifaceted and complex, and its core is the imbalance of calcium homeostasis.
  • the initial mechanical damage will cause the destruction of cell membranes and cytoskeletal components, and increase in internal calcium.
  • Excessive intracellular calcium stimulates the opening of mitochondrial permeability transition pores and increases the permeability of mitochondrial membranes, leading to mitochondrial edema, rupture, and cell apoptosis.
  • Calcium imbalance also causes glutamate toxicity, edema, massive expression of matrix metalloproteinases and the release of inflammatory cytokines. These cascades of events eventually lead to the death of brain parenchymal cells. Therefore, mitochondria play an important role in maintaining the homeostasis of the intracellular environment and cytopathological conditions.
  • mitochondria as the core mediator of the secondary damage cascade, are considered to be effective targets for preventing cell death and dysfunction after TBI.
  • CsA cyclosporine A
  • CsA is a neutral cyclic polypeptide with 11 amino acids isolated from fungi. It has been approved by the FDA as an immunosuppressant and is widely used in organ transplantation. Earlier studies have found that CsA maintains the integrity of mitochondrial function by inhibiting the opening of mitochondrial mPTP, reduces excitotoxicity-mediated mitochondrial calcium uptake and ROS production, and improves the utilization of oxygen by brain tissue after TBI injury. Enter clinical trials for potential neuroprotective drugs.
  • CsA needs to be given a high dose to have neuroprotective effects in the case of oral administration.
  • systemic cyclosporin A levels can produce limiting side effects, such as immunosuppression, liver toxicity and nephrotoxicity, thus limiting its clinical application.
  • the first objective of the present invention is to provide a lipid nano-drug delivery system targeting brain injury lesions. Through the modification of functional penetrating peptides, it has the effect of targeting brain injury lesions and enriching mitochondria. .
  • the second object of the present invention is to provide the application of a lipid nano-drug delivery system targeting brain injury lesions in the preparation of drugs for the treatment of brain injury diseases.
  • the third objective of the present invention is to provide a functional penetrating peptide that modifies the lipid nano-drug delivery system targeting the brain injury focus, and targets the brain injury focus.
  • the fourth objective of the present invention is to provide the application of functional penetrating peptides that modify the lipid nano-delivery system targeting brain injury lesions in preparing the lipid nano-drug delivery system targeting brain injury lesions.
  • the fifth object of the present invention is to provide a method for preparing a lipid nano-drug delivery system targeting a brain injury lesion, wherein the delivery drug is cyclosporin A, and cyclosporin A is prepared by an induced precipitation method.
  • the present invention provides a lipid nano-drug delivery system targeting brain injury lesions, characterized in that the drug delivery system includes lipids, drug delivery and functional penetrating peptides,
  • the functional penetrating peptide is formed by covalently connecting a peptide chain linking the end of a nanocarrier, an arginine-rich penetrating peptide, a matrix metalloproteinase-9 sensitive peptide and a polyanion inhibitory peptide.
  • the sequence of the functional penetrating peptide is the terminal acetylated polypeptide Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 1), Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 2), Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 3), Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 4),
  • the mass ratio of the functional penetrating peptide to lipid is 1:10-1:300, preferably 1:100.
  • the delivery drugs include cyclosporin A, vasoactive peptide, enkephalin, endorphins, neurotensin, and neosandiamine.
  • the delivery drug is cyclosporin A
  • cyclosporin A is prepared by an induced precipitation method.
  • the dilution-induced precipitation technology is used to encapsulate the polypeptide drug cyclosporin A through the core of lipid nanoparticles to repair mitochondria, thereby overcoming the current obstacles of cyclosporin A that are difficult to effectively reach the brain lesions and have a small therapeutic window.
  • the small dose (equivalent to about one-sixteenth of the dose of unmodified CsA) improves the ability to repair cells around the brain injury lesion.
  • the mass ratio of cyclosporin A to lipid is 1:1 to 1:100, preferably 1:4.
  • the present invention provides an application of a lipid nano-drug delivery system targeting brain injury lesions in the preparation of drugs for the treatment of brain injury diseases.
  • the present invention provides a functional penetrating peptide for modifying a lipid nano-delivery system targeting brain injury lesions, characterized in that the sequence of the functional penetrating peptide is terminal Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO.1), Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO.2), GG-FAEKFKEAVK-FAKDY PVGLIG-EGGEGGEGG (SEQ ID NO. 3),
  • the present invention provides the application of functional penetrating peptides that modify the lipid nano-delivery system targeting brain injury lesions in the preparation of a lipid nano-delivery system targeting brain injury lesions .
  • the present invention provides a preparation method of a lipid nano-drug delivery system targeting a brain injury lesion, characterized in that the preparation method comprises a stepwise method or a one-step method, and the stepwise method Including the following steps:
  • the one-step method refers to lipids, cyclosporin A and functional penetrating peptides, which are directly prepared by a microfluidic chip to obtain a functional penetrating peptide modified lipid nano drug delivery system containing cyclosporin A.
  • the step a) induced precipitation method is realized by the microfluidic technology, the precipitation is formed by the water phase and the alcohol phase containing cyclosporin A through the microfluidic chip pipeline, wherein the volume ratio of the alcohol phase to the water phase is preferably 1 :1-1:100, wherein the preferred ratio is 1:8.
  • step b) preparing the cyclosporin A-carrying lipid nano-drug delivery system is prepared by the continuous flow technology of the microfluidic technology, and the lipid-containing phase and the microfluidic induced precipitation method are prepared through the microfluidic pipeline.
  • the cyclosporin A precipitate obtained is self-assembled to prepare a cyclosporin A-carrying lipid nano drug delivery system.
  • the linked nanocarrier end refers to the amino acid sequence connected to the lipid nano drug delivery system, including but not limited to: Ac-FAEKFKEAVKDYFAKFWD-GSG (SEQ ID NO. 9), Ac-FAEKFKEAVKDYFAKFWD-GAGA (SEQ ID NO. 10) Ac-FAEKFKEAVKDYFAKFWD-GG (SEQ ID NO. 11).
  • the arginine-rich penetrating peptide refers to an arginine-rich amino acid sequence, including but not limited to: RRRRRRRRR (SEQ ID NO.12), RRRRRRRRRRRRRRRRRRRRRRRRRRRRR (SEQ ID NO.13).
  • the matrix metalloproteinase-9 sensitive peptide refers to an amino acid sequence that can be specifically hydrolyzed and cleaved by the increased expression of MMP-9 at the brain injury site, including but not limited to: PVGLIG (SEQ ID NO.14), GGGERGPPGPQGAARGFZGTPGL (SEQ ID NO.15), GPLGLLGC (SEQ ID NO.16).
  • the polyanion inhibitory peptide refers to an amino acid sequence rich in acidic amino acids, including but not limited to: EGGEGGEGG (SEQ ID NO. 17), EDDDDK (SEQ ID NO. 18), EEEEEDDDDK (SEQ ID NO. 19).
  • the lipid is lecithin, soybean phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, lysophospholipid, sphingosine, ceramide, sphingomyelin
  • phospholipids cerebrosides, monosialotetrahexose gangliosides and their derivatives.
  • the induced precipitation can be microfluidic technology, reverse microemulsion, and salting out.
  • the microfluidic technology is preferred in this application, in which the water phase and the oil phase containing cyclosporin A pass through the microfluidic chip pipeline to form a precipitate.
  • the present invention adopts lipid nano-carriers and utilizes the core technology of dilution-induced precipitation to encapsulate CsA to realize drug delivery to the brain, which can effectively solve the problem that CsA is difficult to dissolve in water and difficult to cross the blood-brain barrier.
  • the present invention further designs a functional penetrating peptide sensitive to matrix metalloproteinase-9 to modify the CsA-loaded lipid nanocarrier.
  • the functional penetrating peptide is composed of the following parts: the alpha helix peptide at the front end connects the nanocarrier and the functional penetrating peptide; then the arginine-rich penetrating peptide composed of multiple arginines brings the nanocarrier into the cell ; Because of the high positiveness of arginine oligopeptides, it lacks specificity to cells, so a matrix metalloproteinase-9 sensitive peptide was designed to be connected after the penetrating peptide, aiming to specifically increase the expression of the brain injury site The matrix metalloproteinase-9 hydrolyzes and cleaves and exposes the arginine-rich penetrating peptide, which then brings the nano into the cells of the damaged part, making it brain-damage-targeting; finally, a polyanion inhibitory peptide is connected to pass the positive The negative charge function keeps the positive charge of the arginine oligopeptide in a shielded state during the cycle and
  • the advantage of the present invention is that by modifying the functional penetrating peptide, it can target the brain injury lesion and achieve the effect of mitochondrial enrichment.
  • the dilution-induced precipitation technology is used to encapsulate the polypeptide drug cyclosporin A through the core of lipid nanoparticles to repair mitochondria, thereby overcoming the current obstacles of cyclosporin A that are difficult to effectively reach the brain lesions and have a small therapeutic window.
  • the small dose (equivalent to about one-sixteenth of the dose of unmodified CsA) improves the ability to repair cells around the brain injury lesion.
  • Figure 1A is a transmission electron microscope observation of cyclosporin A
  • B is an electron microscope morphology of liposomes without cyclosporin A, scale bar: 50nm.
  • C is the difference in uptake of astrocytes by liposomes containing different amounts of penetrating peptides.
  • F and G are the influence of anhydrous ethanol phase and water on the nanostructure during preparation.
  • Cyclosporin A lipid nano-delivery system (GCAP) modified with functional penetrating peptide is taken up by primary astrocytes and co-localized with mitochondrial markers. Scale bar: 50 ⁇ m.
  • FIG. 3 The protective effect of cyclosporin A-containing lipid nano-delivery system (GCAP) modified with functional penetrating peptide on microglia (A) and neurons (B) in vitro, *p ⁇ 0.05,** p ⁇ 0.01, ***p ⁇ 0.0001.
  • GCAP cyclosporin A-containing lipid nano-delivery system
  • Figure 4 The in vivo targeting effect of functional penetrating peptide modified lipid nano drug delivery system (GNCP) on brain injury lesions, scale: 400 ⁇ m and 500mm.
  • GNCP functional penetrating peptide modified lipid nano drug delivery system
  • Figure 5 The in vivo pharmacodynamic evaluation of cyclosporin A-containing lipid nano-delivery system (GCAP) modified by functional penetrating peptides on craniocerebral injury, scale: 500 ⁇ m.
  • GCAP cyclosporin A-containing lipid nano-delivery system
  • Fig. 6 The improvement evaluation of the cyclosporin A lipid nano-delivery system (GCAP) modified with functional penetrating peptide on the cognitive function of mice, *p ⁇ 0.05.
  • Fig. 7 Safety evaluation of cyclosporin A-containing lipid nano-delivery system (GCAP) modified with functional penetrating peptide, scale: 100 ⁇ m.
  • GCAP cyclosporin A-containing lipid nano-delivery system
  • Example 1 Preparation and characterization of a lipid nano-drug delivery system containing cyclosporin A modified by functional penetrating peptides
  • the blank liposomes were prepared by membrane hydration method.
  • Weigh 2mg of ganglioside (GM1), add 2ml of chloroform: methanol 2:1 mixture to dissolve, prepare 1mg/mL ganglioside stock solution.
  • lipid such as DMPC
  • CsA cyclosporine A
  • the solid-phase peptide synthesis method was used to synthesize functional peptides Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 1), Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO. 2), Ac -FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO.3),
  • the specific method insert the corresponding amino acid on the chloromethyl polystyrene resin, and remove the amino protecting group under the protection of trifluoroacetic acid. Then it was cut by hydrogen fluoride, precipitated with ether in an ice bath, dissolved in acetonitrile and then evaporated, and further purified with an acetonitrile water system.
  • the lipid nano-delivery system containing cyclosporin A modified with functional penetrating peptide was negatively stained with phosphotungstic acid for 5 minutes, and the morphology was observed by transmission electron microscope at 80kV.
  • the CsA-containing functional penetrating peptide-modified lipid nano-delivery system (A) has a more round appearance than the unloaded functional penetrating peptide-modified lipid nano-delivery system (B)
  • the particle size is obviously increased and uniform, and the dispersibility is good, indicating that the CsA drug has been successfully encapsulated.
  • cyclosporin A with the ratio of penetrating peptide (SEQ ID NO.1) and lipid of 1:5, 1:10, 1:100, 1:300 and 1:1000 in step (1) of this example Lipid nano drug delivery system, and add red fluorescent label DiI (accounting for 1% of the lipid mass) when the lipid film is formed.
  • the cyclosporin to lipid ratios were 1:0.5, 1:1, 1:4, 1:20, 1:100 and 1:300 to prepare cyclosporin A lipids
  • the laser particle size analyzer analyzes the particle size of each square, and the results show that 1:1-1:100 are nanostructures with smaller particle sizes ( Figure 1D).
  • the encapsulation efficiency of cyclosporine A was determined by high performance liquid chromatography. The results showed that 1:1-1:300 all showed good encapsulation efficiency (Figure 1E), but at 1:300, the drug loading was only about 0.27%. The low drug loading makes it difficult for the formula here to achieve sufficient drug concentration in subsequent in vivo experiments, so 1:1-1:100 is preferred as the dominant ratio, among which 1:4 is the best.
  • the volume ratio of the absolute ethanol phase containing cyclosporin to the aqueous phase of the liposome solution is set to 1:0.5. 1:1,1:4,1:20,1:8,1:100 and 1:300, the laser particle size analyzer analyzes the particle size and particle size polydispersity coefficient (PDI) of each square, the result shows 1:1- At 1:100, a smaller and uniform nanostructure (as shown in Figure 1F, G) is prepared, and 1:8 is the optimal ratio.
  • PDI particle size polydispersity coefficient
  • Example 2 The lipid nano-delivery system of cyclosporin A modified with functional penetrating peptide is taken up by primary astrocytes and co-localized with mitochondria
  • Example 1 The same as in Example 1, the thin film hydration method was used to prepare drug-loaded common liposomes.
  • the fluorescent dye DiI (20-100 ⁇ g) was added to a 500ml round-bottomed flask, and then the functional transmembrane was prepared as in Example 1.
  • Peptide modified cyclosporin A lipid nano-delivery system adding 1 mg/ml functional penetrating peptide (SEQ ID NO.3) to the CsA-loaded solid according to the ratio of 1/30 and 1/100 of the total lipid weight In the liposome of the inner core, place it in a shaker at 200 rpm and incubate overnight at 4°C to obtain a fluorescently labeled functional penetrating peptide modified cyclosporin A lipid nano-delivery system, and evaluate different ratios of functional penetrating peptides. The influence of membrane peptides on the targeting effect.
  • Example 3 In vitro functional penetrating peptide modified lipid nano-delivery system containing cyclosporin A protects microglia and neurons
  • the microfluidic technology is used to optimize the preparation of a lipid nano-drug delivery system containing cyclosporin A modified with functional penetrating peptide (SEQ ID NO. 8).
  • a lipid nano-drug delivery system containing cyclosporin A modified with functional penetrating peptide (SEQ ID NO. 8).
  • Phase 10ml set the flow rate ratio, enter the microfluidic chip at a total flow rate of 5ml/min, to obtain a solution of the lipid nano-drug delivery system containing functional penetrating peptide modified cyclosporin A, which is replaced by dialysis Filter after the aqueous solution.
  • BV2 cells Culture microglia (BV2 cells) in a 96-well plate. Incubate for 2h under hypoxia (37°C, 5% CO 2 , 95% N 2 ), and change the medium to Earle's balanced salt solution without glucose. After 2 hours, the normal neuron culture medium was replaced, and the oxygen-glucose deprivation cell model was established to simulate the state of cells in the brain after head injury.
  • NMDA 100 ⁇ mol/L
  • glycine 10 ⁇ mol/L
  • glycine 10 ⁇ mol/L
  • the cytotoxicity rating test CCK-8 was used to investigate the effect of CsA and GCAP on the restoration of cell viability after 3 hours of administration. After 3 hours of drug treatment at 37°C, add 10 ⁇ l CCK-8, 450nm wavelength microplate reader to read the value.
  • GCAP significantly stabilizes the mitochondrial membrane potential at low concentrations and can effectively restore the cell viability of damaged neurons, indicating that the functional penetrating peptide modified cyclosporin A lipid nanodelivery
  • the drug system has a protective effect on microglia and neurons.
  • Example 4 In vivo targeting effect of functional penetrating peptide modified lipid nano-delivery system (GNCP) on brain injury lesions
  • GNCP functional penetrating peptide modified lipid nano-delivery system
  • Example 2 a lipid nano-drug delivery system modified with a functional penetrating peptide (SEQ ID NO. 1) carrying fluorescently labeled DiR was prepared.
  • CCI mouse controlled cortical impact model
  • GNCP small animal in vivo imaging experiment to observe the distribution of GNCP in vivo: 8 C57BL/6 male healthy mice were divided into administration group and sham operation group according to the random number table method, and the function of DiR labeling was injected into the tail vein on the 7th day after CCI. Lipid nanocarriers modified by sex-penetrating peptides.
  • the dosing schedule is as follows: Dosing group: GNCP, GNC, (dose of DMPC 5mg/kg), sham operation group; 3 hours after each group of mice are administered, they are anesthetized to death, and the heart, liver, spleen, lungs, and lungs are taken out. Kidney and intact brain tissue. After rinsing with normal saline, it is placed on a small animal in vivo imager to collect images.
  • a cryostat was used to make continuous coronal sections of the brain tissue with a thickness of 20 ⁇ m. Place the sections to dry at room temperature. After rinsing with PBS, incubate the stained nucleus with 100ng/mL DAPI for 30 min in the dark, rinse with PBS, wipe off water stains, mount the slides with mounting solution, and store in the dark.
  • the laser confocal microscope collects slice data, and the excitation light wavelength is 405nm and 568nm.
  • the results are shown in Figure 4.
  • the GNCP preparation containing functional penetrating peptide is more distributed in the brain injury site (A), and less in the surrounding main organs (B) .
  • the amount of fluorescence of GNCP in the brain parenchyma on the injured side was significantly higher than that of GNC, indicating that GNCP nanocarriers were more concentrated in the injury site (C).
  • Whole brain slices further confirmed the enrichment of GNCP in brain injury lesions (D).
  • Example 5 In vivo pharmacodynamic evaluation of cyclosporin A-carrying lipid nano-delivery system (GCAP) modified by functional penetrating peptide on craniocerebral injury
  • the preparation method is the same as in Example 1, and the functional penetrating peptide is SEQ ID NO.1.
  • Glial fibrillary acidic protein (GFAP) antibody immunohistochemical staining was used to show the activation of astrocytes in the brain of CCI model mice after administration. The sections were observed and photographed under a microscope.
  • Nissl staining was used to detect the pathological changes of neurons in the brain of CCI model mice after administration.
  • the paraffin sections were dewaxed and stained with Nissan, stained with toluidine blue staining solution for 3 minutes, and differentiated with 95% alcohol. Finally, the slices were dried in a drying machine and sealed with neutral gum. Observe and take pictures of the slices under a microscope.
  • Microglial cell specific antibody (IBA1) antibody immunohistochemical staining was used to show the activation of microglial cells in the mouse brain after administration. The sections were observed and photographed under a microscope.
  • GCAP effectively reduces the activation of GFAP-labeled astrocytes (A) and the activation of IBA1-labeled microglia (C), and has a significant therapeutic effect on the pathological changes of neurons.
  • the pathological changes of the brain neurons on the injured side were significantly improved compared with the normal saline group, while maintaining the morphology of the hippocampus and cortical neurons of the contralateral normal mouse.
  • the cell arrangement was still scattered and the tissue edema was obvious in the CsA solution group after one week of administration.
  • the CsA drug concentration corresponding to the GCAP drug is 1.26 mg/kg/d, which is only one-sixteenth of the control group with a CsA preparation alone, and the effect is stronger.
  • Example 6 Evaluation of the improvement of the cognitive function of mice by the cyclosporin A lipid nano-delivery system (GCAP) modified by functional penetrating peptide
  • the preparation method is the same as in Example 1, and the functional penetrating peptide is SEQ ID NO.1.
  • CCI model C57BL/6 mice were randomly divided into groups, 6 mice in each group, and they were given tail vein administration as follows for continuous administration for 14 days: sham group (sham): normal saline; injury model group (CCI): Normal saline; CsA solution group: 20mg/kg/d; GNCP group: GNCP solution, 16mg/kg/d (lipid concentration); GCAP group: GCAP solution, 16mg/kg/d (lipid concentration, corresponding The concentration of CsA is 1.26mg/kg/d).
  • the Morris water maze was used for behavioral training and testing of mice in each group of mice 14 days after the administration.
  • the water maze consists of three parts: a circular pool, a platform and a recording system.
  • the circular pool has a diameter of 150cm and a height of 50cm.
  • the pool is divided into 4 quadrants of I, II, III and IV.
  • the pool is filled with water 30cm deep.
  • White edible titanium dioxide is added to make the water opaque.
  • the water temperature is kept at about 25°C. Spatial reference objects and positions are set around the pool Keep unchanged (doors, cameras, signs on the wall, etc.) for mice to locate and learn and remember the position of the platform.
  • the cylindrical transparent platform has a diameter of 9cm and a height of 28cm.
  • mice started on the 9th day of continuous administration and lasted for 5 days.
  • the daily training is based on the principle of random arrangement.
  • the mice are placed into the water from the water entry points of quadrants I, II, III, and IV.
  • the order of entering the water is different every two days.
  • the computer video analysis system monitors and records the mice’s movement in real time.
  • the entry point starts swimming trajectory in the water and the time required to find the platform (incubation period), etc.
  • Each mouse receives 4 trainings a day, and the incubation period is set to 90s for each training. If the mouse does not find a platform within 90s, it needs to be led to the platform and stayed for 30s. At this time, the incubation period is recorded as 90s.
  • the results are shown in Figure 6.
  • the results of the positioning navigation experiment show that the sham operation group and the GCAP group can find the platform faster than the other four groups from the 2nd day (A), the number of times the mice in the space exploration experiment (B) ), the percentage of staying in the target platform quadrant (C) and swimming trajectory (D) show that the search path of mice in the sham operation group and GCAP group is closer to the platform position. It shows that GCAP can significantly improve the spatial learning and memory ability of brain injury model CCI mice.
  • the preparation method is the same as in Example 1, and the functional penetrating peptide is SEQ ID NO.1.
  • Embodiment 8 Microfluidic control preparation method
  • Two-step method Weigh 7.2 mg of DMPC and 0.5 mg of cyclosporine A, add 1 ml of absolute ethanol to dissolve, and prepare the alcohol phase. 7ml ultrapure water is the water phase.
  • the cyclosporin A-loaded nano-lipid solution was prepared through the microfluidic chip with the volume ratio of ethanol:water of 1:7.
  • the obtained lipid nano solution is added with the penetrating peptide aqueous solution, and the cyclosporin A-containing MMP sensitive lipid nano drug delivery system is assembled through the microfluidic system at a volume ratio of 1:1 (prescription 1).
  • the prepared prescription 1 was used to remove the ethanol in the solvent with an ultrafiltration centrifuge tube with a molecular weight cut-off of 10kDa, and resuspended to 2ml with ultrapure water. Zetasizer was used to measure the light scattering particle size and Zeta potential.
  • One-step method Weigh 7.2 mg of DMPC and 0.5 mg of cyclosporine A, add 1 ml of absolute ethanol to dissolve, and prepare the alcohol phase. Take 40 ⁇ l 1mg/ml penetrating peptide aqueous solution and dissolve it in 6960 ⁇ l ultrapure water as the water phase (total volume 7ml).
  • the cyclosporin A-containing MMP-sensitive lipid nano-drug delivery system was prepared through the microfluidic chip with a volume ratio of ethanol:water of 1:7 (prescription 2).
  • the prepared prescription 1 was used to remove the ethanol in the solvent with an ultrafiltration centrifuge tube with a molecular weight cut-off of 10kDa, and resuspended to 2ml with ultrapure water. Zetasizer was used to measure the light scattering particle size and Zeta potential.
  • the solid-phase peptide synthesis method is used to synthesize functional peptides.
  • the specific method insert the corresponding amino acid on the chloromethyl polystyrene resin, and remove the amino protecting group under the protection of trifluoroacetic acid. Then it was cut by hydrogen fluoride, ether was precipitated in an ice bath, acetonitrile was dissolved and then revolved, and the acetonitrile water system was used for further purification.
  • a lipid nano-delivery system containing cyclosporin A with a 1:100 ratio of penetrating peptide to lipid was prepared in the same way as in Example 8 (see Table 1), and the red fluorescent label DiI was added when the lipid film was formed (Accounting for 1% of lipid mass).
  • the functional peptides are the end-group acetylated alpha helix penetrating peptide Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ ID NO.
  • the laser particle size analyzer measures its particle size and surface potential.
  • Culture primary astrocytes in 96-well plates add the above lipid nano-delivery system containing different transmembrane peptides in the presence of MMP-9 (500ngmL-1), incubate at 37°C for 3 hours, fix with 3.7% formaldehyde After 10 minutes, DAPI stains the nucleus, the high-content drug analysis system analyzes the amount of each preparation taken by the cells. The results show that it contains functional penetrating peptides (sequences SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7. SEQ ID NO. 8) lipid nano-drug delivery system is taken up by cells significantly higher than the unmodified functional penetrating peptide prescription.

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Abstract

一种靶向颅脑损伤病灶的脂质纳米递药系统及其制备方法和应用，所述递药系统包括脂质、递送药物和功能性穿膜肽，所述功能性穿膜肽由链接纳米载体端、富精氨酸穿膜肽、基质金属蛋白酶-9敏感肽和聚阴离子抑制肽的肽链共价连接形成。所述脂质纳米递药系统通过功能性穿膜肽的修饰，能够靶向颅脑损伤病灶并实现线粒体富集的效果。利用稀释诱导沉淀技术通过脂质纳米粒核心包载多肽类药物环孢素A，进行线粒体的修复，由此克服目前的环孢素A难以有效达到颅脑病灶并治疗窗口小的阻碍，以较小的给药剂量提高了对颅脑损伤病灶周围细胞修复的能力。

Description

靶向颅脑损伤病灶的脂质纳米递药系统及其制备方法和应用 技术领域
本发明涉及技术纳米生物医学材料技术-纳米药物和药物传递系统领域，尤其涉及一种靶向颅脑损伤病灶的脂质纳米递药系统及其制备方法和应用。
背景技术
创伤性颅脑损伤(Traumatic brain injury,TBI)是神经外科中常见的疾病，其较高的死亡率和致残率给患者家庭带来极大的经济和心理负担。目前认为TBI的损伤可分为原发性和继发性两类，原发性损伤主要发生于外力作用点，由机械损伤引起的脑实质损伤。然而大脑受到的损害不仅发生在损伤瞬间，早期的机械损伤常常诱发继发性损伤级联反应，包括细胞兴奋性毒性、血管源性和细胞毒性水肿、缺氧缺血、线粒体功能障碍、氧化应激和炎症等加剧原受损组织的损害。由于缺乏有效的治疗手段，众多颅脑损伤患者在意识恢复之后遗留下不同程度的神经功能障碍，而如何改善创伤性颅脑损伤引起的神经功能障碍缺少具体治疗方案。
TBI神经功能障碍的病理生理机制是多方面且复杂的，其核心是钙稳态的失调。最初机械性的损伤会引起细胞膜和细胞骨架元件的破坏，内钙增加，过量的细胞内钙刺激线粒体通透性转换孔的开放增加线粒体膜的通透性，导致线粒体水肿，破裂，引起细胞凋亡；钙失调同时也引起谷氨酸毒性、水肿、基质金属蛋白酶大量表达和炎性细胞因子的释放。这些级联事件最终导致脑实质细胞死亡。因此线粒体在维护细胞内环境稳态和细胞病理条件下都起重要的作用。近年来线粒体作为继发损伤级联反应的核心介质，被认为是预防TBI后细胞死亡和功能障碍的有效靶点。
目前已发现多种靶向线粒体的神经保护性药物，其中对环孢素A(cyclosporine A，CsA)的研究比较广泛。CsA是从真菌中分离得到的含11个氨基酸的中性环状多肽，被FDA批准作为免疫抑制剂广泛应用于器官移植中。早前的研究发现，CsA通过抑制线粒体mPTP的开放维持线粒体功能的完整性，减少了兴奋性毒性介导的线粒体钙摄取和ROS的产生，使TBI损伤后脑组织对氧的利用得到提高，被视为具有潜力的神经保护药进入临床试验。然而由于CsA的水溶性差，与血浆蛋白的结合率高，难以通过血脑屏障，且治 疗窗狭窄，口服情况下需要给予高剂量才具有神经保护作用。在高剂量和慢性给药的情况下，全身环孢素A水平会产生限制性的副作用，如免疫抑制、肝毒性和肾毒性，从而限制了其临床应用。
发明内容
本发明的第一个目的在于，提供一种靶向颅脑损伤病灶的脂质纳米递药系统，通过功能性穿膜肽的修饰，起到靶向颅脑损伤病灶并在线粒体富集的效果。
本发明的第二个目的在于，提供靶向颅脑损伤病灶的脂质纳米递药系统在制备治疗颅脑损伤疾病药物中的应用。
本发明的第三个目的在于，提供一种修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽，靶向颅脑损伤病灶。
本发明的第四个目的在于，提供修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽在制备靶向颅脑损伤病灶的脂质纳米递药系统中的应用。
本发明的第五个目的在于，提供靶向颅脑损伤病灶的脂质纳米递药系统的制备方法，其中递送药物为环孢素A，且环孢素A由诱导沉淀方法制备。
为了实现上述第一个目的，本发明提供了一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述递药系统包括脂质、递送药物和功能性穿膜肽，所述功能性穿膜肽由链接纳米载体端、富精氨酸穿膜肽、基质金属蛋白酶-9敏感肽和聚阴离子抑制肽的肽链共价连接形成。
[根据细则91更正 29.01.2021]　
作为一个优选方案，所述功能性穿膜肽的序列为端基乙酰化多肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.1)，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.2)，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.3)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.4)，
AC-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK(SEQ ID NO.5)， AC-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK(SEQ ID NO.6)，AC-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG(SEQ ID NO.7)，AC-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGEGG(SEQ ID NO.8)中的任一所示。
作为一个优选方案，所述功能性穿膜肽与脂质的质量比为1:10-1:300，优选1:100。
作为一个优选方案，所述递送药物包括环孢素A、血管活性肽、脑啡肽、内啡肽、神经降压肽和新山地明。
作为进一步的优选方案，所述递送药物为环孢素A，且环孢素A由诱导沉淀方法制备。利用稀释诱导沉淀技术通过脂质纳米粒核心包载多肽类药物环孢素A，进行线粒体的修复，由此克服目前的环孢素A难以有效达到颅脑病灶并治疗窗口小的阻碍，以较小的给药剂量(相当于约十六分之一无修饰CsA的给药剂量)提高了对颅脑损伤病灶周围细胞修复的能力。
作为进一步的优选方案，所述环孢素A与脂质的质量比为1:1-1:100，优选为1:4。
为了实现上述第二个目的，本发明提供了一种靶向颅脑损伤病灶的脂质纳米递药系统在制备治疗颅脑损伤疾病药物中的应用。
[根据细则91更正 29.01.2021]　
为了实现上述第三个目的，本发明提供了一种修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽，其特征在于，所述功能性穿膜肽的序列为端基乙酰化多肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.1)，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.2)，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.3)，
[根据细则91更正 29.01.2021]　
Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.4)，
[根据细则91更正 29.01.2021]　
Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK(SEQ ID NO.5)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK(SEQ ID NO.6)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG(SEQ ID NO.7)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGEGG(SEQ ID NO.8)。
为了实现上述第四个目的，本发明提供了修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽在制备靶向颅脑损伤病灶的脂质纳米递药系统中的应用。
为了实现上述第五个目的，本发明提供了靶向颅脑损伤病灶的脂质纳米递药系统的制备方法，其特征在于，所述制备方法包括分步法或者一步法，所述分步法包括如下步骤：
a)采用稀释诱导沉淀方法形成环孢素A的沉淀溶液；
b)制备载环孢素A的脂质纳米递药系统；
c)通过在上述b)制备的纳米递药系统溶液中加入所述的功能性穿膜肽，制备得到功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统。
所述一步法是指脂质、环孢素A和功能性穿膜肽，直接通过微流控芯片制备得到功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统。
作为一个优选方案，步骤a)诱导沉淀方法由微流控技术实现，通过水相和含有环孢素A的醇相通过微流控芯片管道形成沉淀，其中醇相与水相体积比优选为1:1-1:100，其中优选比例为1:8。
作为一个优选方案，步骤b)制备载环孢素A的脂质纳米递药系统采用微流控技术的连续流技术制备，通过微流控管道将含脂质相与微流控诱导沉淀方法制得的环孢素A沉淀通过自组装，制得载环孢素A的脂质纳米递药系统。
[根据细则91更正 29.01.2021]　
所述链接纳米载体端是指为模拟与脂质纳米递药系统相连的氨基酸序列，包括但不限于：Ac-FAEKFKEAVKDYFAKFWD-GSG(SEQ ID NO.9)、Ac-FAEKFKEAVKDYFAKFWD-GAGA(SEQ ID NO.10)、 Ac-FAEKFKEAVKDYFAKFWD-GG(SEQ ID NO.11)。
所述富精氨酸穿膜肽是指富含精氨酸的氨基酸序列，包括但不限于：RRRRRRRRR(SEQ ID NO.12)、RRRRRRRRRRRRRRRRRR(SEQ ID NO.13)。
所述基质金属蛋白酶-9敏感肽是指为可在脑损伤部位特异性地被表达量增高的MMP-9水解剪切的氨基酸序列，包括但不限于：PVGLIG(SEQ ID NO.14)、GGGERGPPGPQGAARGFZGTPGL(SEQ ID NO.15)、GPLGLLGC(SEQ ID NO.16)。
所述聚阴离子抑制肽是指为富含酸性氨基酸的氨基酸序列，包括但不限于：EGGEGGEGG(SEQ ID NO.17)、EDDDDK(SEQ ID NO.18)、EEEEEDDDDK(SEQ ID NO.19)。
所述脂质为卵磷脂、大豆磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇、磷脂酸、心磷脂、溶血磷脂、鞘氨醇、神经酰胺、鞘磷脂、脑苷脂、单唾液酸四己糖神经节苷脂及其衍生物中的一种或多种。
所述诱导沉淀可以是微流控技术、反向微乳、盐析。本申请中优选微流控技术，通过水相和含有环孢素A的油相通过微流控芯片管道形成沉淀。
本发明采用脂质纳米载体，利用稀释诱导沉淀的技术核心包载CsA实现脑部递药，可以有效地解决CsA难溶于水，且难跨血脑屏障的问题。为实现纳米载体对颅脑损伤部位的靶向递送，本发明进一步设计了具基质金属蛋白酶-9敏感的功能性穿膜肽以修饰载CsA的脂质纳米载体。该功能性穿膜肽由以下几个部分构成：前端的α螺旋肽连接纳米载体与功能性穿膜肽；随后多个精氨酸组成的富精氨酸穿膜肽，将纳米载体带进细胞；由于精氨酸寡肽的高正电性使其对细胞缺乏特异性，因此在穿膜肽后设计连接一段基质金属蛋白酶-9敏感肽，旨在脑损伤部位特异性地被表达量增高的基质金属蛋白酶-9水解剪切并暴露出富精氨酸穿膜肽，进而将纳米带进受损部位的细胞内，使其具脑损伤靶向性；最后连接一段聚阴离子抑制肽，通过正负电荷作用，使精氨酸寡肽的正电荷在循环过程中保持被屏蔽的状态，增强功能性穿膜肽的稳定性。
本发明的优点在于，通过功能性穿膜肽的修饰，能够靶向颅脑损伤病灶并实现线粒体富集的效果。利用稀释诱导沉淀技术通过脂质纳米粒核心包载多肽类药物环孢素A，进行线粒体的修复，由此克服目前的环孢素A难以有效达到 颅脑病灶并治疗窗口小的阻碍，以较小的给药剂量(相当于约十六分之一无修饰CsA的给药剂量)提高了对颅脑损伤病灶周围细胞修复的能力。
附图说明
图1A为透射电镜观察载环孢素A，B为未载环孢素A的脂质体电镜形态，标尺：50nm。C为含不同量穿膜肽的脂质体的星形胶质细胞摄取差异。D和E环孢素A投入比例对载环孢素A的脂质纳米递药系统纳米结构和载药量的影响。F和G为制备时无水乙醇相和水相对纳米结构的影响。
图2功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)被原代星形胶质细胞摄取，且与线粒体标记物共定位。标尺：50μm。
图3功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)对小胶质细胞(A)和神经元(B)的体外保护作用，*p<0.05,**p<0.01，***p<0.0001。
图4功能性穿膜肽修饰的脂质纳米递药系统(GNCP)对颅脑损伤病灶的体内靶向效果，标尺：400μm和500mm。
图5功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)对颅脑损伤的体内药效学评价，标尺：500μm。
图6功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)对小鼠认知功能的改善评价，*p<0.05。
图7功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)的安全性评价，标尺：100μm。
具体实施方式
以下，结合具体实施方式对本发明的技术进行详细描述。应当知道的是，以下具体实施方式仅用于帮助本领域技术人员理解本发明，而非对本发明的限制。
实施例1.功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统的制备和表征
(1)制备
采用薄膜水化法制备空白脂质体。称取2mg神经节苷脂(GM1)，加入2ml氯仿:甲醇＝2:1的混合液溶解，制备成1mg/mL神经节苷脂的储备液。称取3.6mg脂质(如DMPC)于500ml圆底烧瓶中，加入400μl神经节苷脂储备 液，再加入3mL氯仿，置旋转蒸发仪上抽真空1h。称取1mg、2mg及4mg不同质量的环孢素A(CsA)，加入2ml-4ml无水乙醇溶解，制备成1mg/ml CsA的储备液。吸取40μl-4ml CsA储备液，超纯水稀释至4ml，制备得到CsA多肽沉淀液。在上述含脂质薄膜的圆底烧瓶中加入上述制备的CsA多肽沉淀液水化脂质，并置于摇床200rpm，37℃振摇2h至薄膜水化脱落得到载CsA固体内核的脂质体，水浴探头超声(1％功率，5min)进一步减小脂质体粒径。用0.22μm亲水PTFE针式滤器分离过滤除去GCA中未包载进脂质体的CsA多肽沉淀颗粒。按照总脂重量的1/100将1mg/ml功能性穿膜肽加入至载CsA固体内核的脂质体中，置于摇床200rpm，4℃孵育过夜，制备得到载CsA固体内核的功能性多肽修饰的脂质体(GCAP)。
[根据细则91更正 29.01.2021]　
采用固相多肽合成法，合成功能性多肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.1)，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.2)，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.3)，
[根据细则91更正 29.01.2021]　
Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.4)，
[根据细则91更正 29.01.2021]　
Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK(SEQ ID NO.5)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK(SEQ ID NO.6)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG(SEQ ID NO.7)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGEGG(SEQ ID NO.8)。
具体方法：在氯甲基聚苯乙烯树脂上接入相应的氨基酸，在三氟乙酸的保护下脱氨基保护基团。而后通过氟化氢进行切割，乙醚冰浴沉淀，乙腈溶解后 旋蒸，并采用乙腈水体系进一步纯化。
(2)表征
功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统磷钨酸负染5分钟，透射电镜80kV观察形态。如图1，在电镜下含CsA的功能性穿膜肽修饰的脂质纳米递药系统(A)较未载药的功能性穿膜肽修饰的脂质纳米递药系统(B)外观更加圆整，粒径明显增大且均一，分散性良好，说明CsA药物被成功包载。
(3)穿膜肽与脂质比例的筛选
同本实施例步骤(1)中制备穿膜肽(SEQ ID NO.1)与脂质比例为1:5,1:10,1:100,1:300和1:1000的载环孢素A的脂质纳米递药系统，并在脂质成膜时添加红色荧光标记DiI(占脂质质量1％)。
96孔板培养原代星形胶质细胞，给药上述含不同比例的穿膜肽的脂质纳米递药系统，37℃孵育3小时后，3.7％甲醛固定10分钟，DAPI染核后，高内涵药物分析系统分析细胞摄取各制剂的量，如图1C，星形胶质细胞摄取穿膜肽与脂质比例1:10-1:300的纳米递药系统较多，其中1:100为最优比例。
(4)环孢素A与脂质比例的筛选
同本实施例步骤(1)中制备环孢菌素与脂质比例为1:0.5,1:1,1:4,1:20,1:100和1:300的载环孢素A的脂质纳米递药系统，激光粒度仪分析各处方的粒径，结果显示1:1-1:100为粒径较小的纳米结构(如图1D)。高效液相色谱测定环孢素A包封率，结果显示1:1-1:300均显示较好包封率(如图1E)，但1:300时，载药量只有约0.27％，过低的载药量使此处方在后续的体内实验中难以达到足够的药物浓度，故优选1:1-1:100为优势比例，其中为1:4最优。
(5)无水乙醇相与水相比例的筛选
同本实施例步骤(4)中制备载环孢素A的脂质纳米递药系统时，设置含环孢菌素的无水乙醇相与脂质体溶液水相的体积比例为1:0.5,1:1,1:4,1:20,1:8,1:100和1:300的，激光粒度仪分析各处方的粒径和粒径多分散系数(PDI)，结果显示1:1-1:100时制得粒径较小且均一的纳米结构(如图1F，G)，其中1:8为最优比例。
实施例2.功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统被原代星 形胶质细胞摄取且与线粒体共定位
(1)制备
同实施例1采用薄膜水化法制备载药的普通脂质体，在制备脂质薄膜时加入荧光染料DiI(20-100μg)到500ml圆底烧瓶中，后同实施例1制备功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统，按照总脂重量的1/30及1/100比例将1mg/ml功能性穿膜肽(SEQ ID NO.3)加入至载CsA固体内核的脂质体中，置于摇床200rpm，4℃孵育过夜，得到荧光标记的功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统，并评价不同比例的功能性穿膜肽对靶向效果的影响。
(2)原代星形胶质细胞的提取在超净台内操作。取24h内新生SD大鼠，酒精棉消毒，断颈，沿脑中线将大脑分为两半，剥去脑壳以及脑膜，取出海马组织。显微镜下仔细剥离血管膜和其他附带脑组织。取出的海马组织剪碎，加入消化液在37℃消化10-15min。消化结束后，将消化液移入装有10ml完全培养液的离心管中终止消化，加入20μl DNA酶，轻柔吹打15-20次使细胞分散。随后1500rpm常温离心15min。倾去上清，留沉淀，加入完全培养液，将沉淀吹散混匀制成细胞悬液。显微镜下计数，用完全培养液稀释至合适浓度，种于多聚赖氨酸包被好的96孔板，种植密度为1×10 4细胞/孔。置于5％CO2细胞培养箱，37℃培养。
将原代胶质细胞接种于孔板，将脂质量为20μg/ml的载荧光探针DiI的含不同比例的功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统，孵育3小时后，加入500ng/ml MMP蛋白和线粒体指示剂Mitotracker，PBS清洗一次后，多聚甲醛固定。染核后，将细胞于激光共聚焦显微镜检测其摄取制剂的荧光强度及与线粒体的共定位情况。
结果如图2所示，载荧光探针的功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统与线粒体指示剂Mitotracker高度共定位，表明功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统可有效递送至线粒体。
实施例3.体外功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统对小胶质细胞和神经元的保护作用
(1)制备
采用微流控技术优化制备功能性穿膜肽(SEQ ID NO.8)修饰的载环孢素A的脂质纳米递药系统。分别准备含3.6mg DMPC脂质和0.4mg神经节苷脂的无水乙醇相40μl-4ml，含1mg、2mg及4mg的环孢素A的乙醇4-10ml，以及含功能性穿膜肽的水相10ml，设定流速比，以总流速5ml/min的速度进入微流控芯片，得到含功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统的溶液，通过透析置换为水溶液后过滤。
(2)将小胶质细胞(BV2细胞)培养在96孔板内。缺氧环境下培养2h(37℃,5％CO 2，95％N 2)，将培养基换为不含葡萄糖的Earle’s平衡盐溶液。2h后更换正常的神经元培养液，建立氧糖剥夺细胞模型，模拟颅脑损伤后的脑内细胞状态。给予培养液-DMEM、CsA低浓度组2.5μg/ml，CsA高浓度组12.5μg/ml、功能能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)低浓度给药组2.5μg/ml和GCAP高浓度给药组12.5μg/ml，每个浓度6个复孔，给药3小时。提取线粒体，收集细胞，用PBS洗一遍，用胰酶消化细胞，室温离心5-10分钟收集细胞。用冰浴预冷的PBS轻轻重悬细胞沉淀，取少量细胞用于计数，剩余细胞600g，4℃离心5分钟沉淀细胞。弃上清。加入1-2.5ml线粒体分离试剂或临用前添加了蛋白酶抑制剂(PMSF)的线粒体分离试剂至2000-5000万细胞中，轻轻悬浮细胞，冰浴放置10-15分钟。把细胞悬液转移到玻璃匀浆器中，匀浆10-30下左右。细胞匀浆4℃，600g，离心10分钟。上清转移到另一离心管中，4℃，11,000g，离心10分钟。小心去除上清，沉淀即为分离得到的细胞线粒体。把配制好的JC-1染色工作液用JC-1染色缓冲液稀释5倍。0.9ml 5倍稀释的JC-1染色工作液中加入0.1ml总蛋白量为10-100μg纯化的线粒体。用荧光酶标仪检测：检测JC-1单体时激发光/发射光设置为490nm/530nm；检测JC-1聚合物时，激发光/发射光设置为525nm/590nm。
将NMDA(100μmol/L)和甘氨酸(10μmol/L)溶液作用于原代神经元2h后，神经元的活力产生明显下降，建立NMDA毒性细胞模型。通过细胞毒性评级实验CCK-8考察CsA和GCAP给药3小时后对恢复细胞活力的效果。在37℃药物处理3小时后，加入10μl CCK-8，450nm波长酶标仪读值。
结果如图3所示，GCAP在低浓度时即显著稳定线粒体膜电位水平，且能够有效恢复受损神经元的细胞活力，表明功能性穿膜肽修饰的载环孢素A的脂 质纳米递药系统对小胶质细胞和神经元具有保护作用。
实施例4.功能性穿膜肽修饰的脂质纳米递药系统(GNCP)对颅脑损伤病灶的体内靶向效果
(1)制备
同实施例2制备载荧光标记DiR的功能性穿膜肽(SEQ ID NO.1)修饰的脂质纳米递药系统。
(2)小鼠控制性脑皮质撞击模型(CCI)的建立：1)小鼠经腹腔麻醉(5％水合氯醛，0.1mL/10g)后头部固定在脑立体定位仪上，暴露前囟及右侧顶骨，用骨钻于前囟点后方1mm，矢状缝右侧钻一直径约为4mm的骨孔，去除骨瓣，暴露完整硬脑膜。2)采用CCI脑立体定向撞击仪实施撞击，参数设置如下：撞击头直径3mm，撞击速度3m/s，撞击深度1mm，停留时间85ms。3)调节脑立体定位仪上的操纵臂，当撞击头和脑膜表面紧密贴合发出报警声音，即可点击撞击按钮，完成撞击。4)撞击完成后立即缝合头皮，将小鼠放进动物重症监护仓，保持37℃，等待小鼠复苏。假手术组(Sham)除不予实施撞击过程，其余采用相同的麻醉及手术操作程序。
小动物活体成像实验观察GNCP体内分布：取8只C57BL/6雄性健康小鼠按随机数字表法分为给药组和假手术组，分别于CCI术后第7天尾静脉注射DiR标记的功能性穿膜肽修饰的脂质纳米载体。给药方案如下：给药组：GNCP、GNC、(给药剂量均为DMPC 5mg/kg)，假手术组；每组小鼠给药3h后，麻醉处死，取出心、肝、脾、肺、肾和完整脑组织。生理盐水冲洗后置于小动物活体成像仪，采集图像。
冰冻切片观察功能性穿膜肽修饰的脂质纳米载体在脑组织的分布：C57BL/6雄性健康小鼠随机为给药组和假手术组，分别于CCI术后第7天尾静脉注射荧光标记的GNCP载体。给药方案同上。给药3h后，麻醉处死，取出完整大脑，置于4％多聚甲醛中，4℃固定24h。固定后的脑组织依次置于15％和30％的蔗糖-PBS中进行梯度脱水至下沉，脱水后脑组织使用组织包埋液(O.C.T)包埋并置于–20℃凝固。使用冰冻切片机对脑组织作连续冠状切片，厚度20μm。切片置于室温晾干，PBS漂洗后用100ng/mL DAPI避光孵育染核30min，PBS漂洗，拭去水渍，用封片液封片，避光保存。激光共聚焦 显微镜采集切片数据，激发光波长为405nm和568nm。
结果如图4所示，含功能性穿膜肽的GNCP制剂较不含功能性穿膜肽的GCP更多的分布于脑损伤部位(A)，且在周围主要脏器分布较少(B)。GNCP在损伤侧脑实质中的荧光量明显高于GNC，表明GNCP纳米载体在损伤部位更加聚集(C)，通过全脑切片更进一步证实了GNCP在脑损伤病灶的富集(D)。
实施例5.功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)对颅脑损伤的体内药效学评价
(1)制备
制备方法同实施例1，功能性穿膜肽为SEQ ID NO.1。
(2)将CCI模型C57BL/6小鼠假手术组(sham)：给予生理盐水；损伤模型组(CCI)：给予生理盐水；CsA溶液组：20mg/kg/d；GNCP组：给予GNCP溶液，16mg/kg/d(脂质浓度)；GCAP组：给予GCAP溶液，16mg/kg/d(脂质浓度，对应CsA浓度为1.26mg/kg/d)。每组3只，连续给药7天。7天后麻醉小鼠，0.9％生理盐水灌流后再以4％多聚甲醛溶液灌流固定，取出完整大脑，置于4％多聚甲醛溶液中4℃固定过夜，浸蜡，石蜡包埋，切片，切片厚度5μm。
采用胶质纤维酸性蛋白(GFAP)抗体免疫组化染色显示给药后CCI模型小鼠脑内星形胶质细胞激活的情况，显微镜下对切片进行观测并拍照。
采用尼氏染色法(Nissl staining)检测给药后CCI模型小鼠脑内神经元的病理改变。石蜡切片经脱蜡后尼式染色，甲苯胺蓝染液染色3min，95％酒精分化，最后烘片机烤干，中性树胶封片。显微镜下对切片进行观测并拍照。
采用小胶质细胞特异抗体(IBA1)抗体免疫组化染色显示给药后小鼠脑内小胶质细胞激活的情况，显微镜下对切片进行观测并拍照。
结果如图5所示，GCAP有效降低GFAP标记的星形胶质细胞的激活(A)以及IBA1标记的小胶质细胞的激活(C)，同时对于神经元的病理性改变有明显的治疗作用，损伤侧大脑神经元的病理性改变较生理盐水组明显改善，同时维持了对侧正常小鼠海马以及皮层神经元的形态，而CsA溶液组给药周后，细胞排列仍散乱，组织水肿明显，显示对神经元的病理性改变没有明显的抑制和修复作用(B)。GCAP药物所对应的CsA药物浓度为1.26mg/kg/d，仅为对 照单纯CsA制剂组的十六分之一，而疗效更强。
实施例6.功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)对小鼠认知功能的改善评价
(1)制备
制备方法同实施例1，功能性穿膜肽为SEQ ID NO.1。
(2)CCI模型C57BL/6小鼠随机分组，每组6只，按如下方式进行尾静脉给药，连续给药14d：假手术组(sham)：给予生理盐水；损伤模型组(CCI)：给予生理盐水；CsA溶液组：20mg/kg/d；GNCP组：给予GNCP溶液，16mg/kg/d(脂质浓度)；GCAP组：给予GCAP溶液，16mg/kg/d(脂质浓度，对应CsA浓度为1.26mg/kg/d)。
Morris水迷宫实验：
每组小鼠给药14天后采用Morris水迷宫对小鼠进行行为学训练及测试。水迷宫由圆形水池、平台和记录系统三部分组成。圆形水池直径150cm，高50cm，水池分为Ⅰ、Ⅱ、Ⅲ和Ⅳ4个象限，水池注水30cm深，加入白色食用二氧化钛使水体不透明，水温保持在25℃左右，水池四周设空间参照物且位置保持不变(门、摄像头及墙上标志等)，以供小鼠定位以及学习记忆平台位置。圆柱形透明平台直径9cm，高在28cm，包裹白布放置于Ⅳ象限，平面没于水面下2cm。摄像头置于水池中央上方，采用Morris水迷宫视频分析系统2.0监测并记录小鼠的游泳轨迹。行为学测试过程中保持室内安静、光线柔和一致、各参照物位置不变。
定位航行实验：小鼠连续给药第9天开始，历时5天。每天训练按随机排列原则分别从Ⅰ、Ⅱ、Ⅲ、Ⅳ象限的入水点将小鼠面向池壁放入水中，每相邻两天的入水顺序不同，计算机视频分析系统实时监测并记录小鼠从入水点开始在水中游泳轨迹以及找到平台所需时间(潜伏期)等。每只小鼠每天接受4次训练，每次训练设定潜伏期为90s，如若小鼠90s内未找到平台，需将其引领至平台，并停留30s，这时潜伏期记为90s。
空间探索实验：5天定位航行试验后，于第6天撤去平台，分别从Ⅰ、Ⅲ象限入水点将小鼠面向池壁放入水中，记录小鼠在60s内在平台所在象限区域的时间百分比以及小鼠搜索平台的轨迹和跨越平台的次数。
结果如图6所示，定位航行实验结果显示假手术组和GCAP组从第2d开始较其他四组能更快的找到平台(A)，在空间探索实验中的小鼠的穿台次数(B)、停留目标平台象限时间百分比(C)和游泳轨迹(D)显示，假手术组和GCAP组小鼠的探寻路径更接近于平台位置。表明GCAP对脑损伤模型CCI小鼠空间学习记忆能力具有明显改善作用。
实施例7.功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统(GCAP)的安全性评价
(1)制备
制备方法同实施例1，功能性穿膜肽为SEQ ID NO.1。
(2)在Morris水迷宫实验结束后，取小鼠的心、肝、脾、肺以及肾，制备成蜡块并切片。对组织器官的切片进行HE染色，显微镜下观察并拍照。初步评价GCAP体内安全性。
结果如图7所示，在纳米载体易聚集的肝、脾组织中，肝细胞无浊肿，无小叶内炎症，脾白髓及红髓结构清晰，未出现显微镜下可见的形态学变化；在分布较少的心、肺组织中，心肌纤维未见异常，肺组织中肺泡结构正常。肾组织中，肾小球及肾小管结构正常，未表现出显微镜下可见的形态学变化。结果表明CCI模型小鼠尾静脉连续给药GCAP在14天后对外周主要组织器官中无明显损伤。
实施例8.微流控制备方法
两步法：称取7.2mg DMPC和0.5mg环孢素A，加入1ml无水乙醇溶解，制备成醇相。7ml超纯水为水相。以乙醇:水体积比1:7通过微流控芯片，制备得到载有环孢素A的纳米脂质溶液。得到的脂质纳米溶液再加穿膜肽水溶液，以1:1体积比通过微流控系统组装成载环孢素A的MMP敏感脂质纳米递药系统(处方1)。将制备得到处方1用截留分子量为10kDa的超滤离心管将溶剂中的乙醇除去，用超纯水重悬至2ml。采用Zetasizer测定光散射粒径和Zeta电位。
一步法：称取7.2mg DMPC和0.5mg环孢素A，加入1ml无水乙醇溶解，制备成醇相。取40μl 1mg/ml穿膜肽水溶液，溶于6960μl超纯水中为水相(总体积7ml)。以乙醇:水体积比1:7通过微流控芯片，制备得到载环孢素 A的MMP敏感脂质纳米递药系统(处方2)。将制备得到处方1用截留分子量为10kDa的超滤离心管将溶剂中的乙醇除去，用超纯水重悬至2ml。采用Zetasizer测定光散射粒径和Zeta电位。
结果发现，处方1、处方2粒径分别为28.03±2.35nm和29.69±1.92nm，多分散系数PDI分别为0.13和0.23，zeta电位分别为-8.26±0.27和-1.36±0.74mV，表明不管采用两步法或一步法微流控制备技术对所得制剂的粒径及其分布影响不大。
实施例9.功能性穿膜肽的优化
采用固相多肽合成法，合成功能性多肽。具体方法：在氯甲基聚苯乙烯树脂上接入相应的氨基酸，在三氟乙酸的保护下脱氨基保护基团。而后通过氟化氢进行切割，乙醚冰浴沉淀，乙腈溶解后旋蒸，并采用乙腈水体系进一步纯化。
[根据细则91更正 29.01.2021]　
同本实施例8一步法制备穿膜肽与脂质比例为1:100的载环孢素A的脂质纳米递药系统(如表1)，并在脂质成膜时添加红色荧光标记DiI(占脂质质量1％)。功能性多肽为端基乙酰化α螺旋穿膜肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.1)，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.2)，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.3)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG(SEQ ID NO.4)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK(SEQ ID NO.5)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK(SEQ ID NO.6)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG(SEQ ID NO.7)，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGE GG(SEQ ID NO.8)和Ac-FAEKFKEAVKDYFAKFWD-GSG(SEQ ID NO.9)、Ac-FAEKFKEAVKDYFAKFWD-GAGA(SEQ ID NO.10)、Ac-FAEKFKEAVKDYFAKFWD-GG(SEQ ID NO.11)、RRRRRRRRR(SEQ ID NO.12)、RRRRRRRRRRRRRRRRRR(SEQ ID NO.13)、PVGLIG(SEQ ID NO.14)、GGGERGPPGPQGAARGFZGTPGL(SEQ ID NO.15)、GPLGLLGC(SEQ ID NO.16)、EGGEGGEGG(SEQ ID NO.17)、EDDDDK(SEQ ID NO.18)、EEEEEDDDDK(SEQ ID NO.19)、Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GERGPPGPQGAARGFZGTPGL-EGGEGGEGG(SEQ ID NO.20)、Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR(SEQ ID NO.21)、Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRRRRRRRRRRR(SEQ ID NO.22)和Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRRRRRRRRRRR-PVGLIG(SEQ ID NO.23)。
激光粒度仪测定其粒径和表面电位。96孔板培养原代星形胶质细胞，在MMP-9(500ngmL-1)存在下分别加入上述含不同穿膜肽的脂质纳米递药系统，37℃孵育3小时后，3.7％甲醛固定10分钟，DAPI染核后，高内涵药物分析系统分析细胞摄取各制剂的量。结果显示，含功能性穿膜肽(序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8)的脂质纳米递药系统被细胞摄取量均显著高于未修饰功能性穿膜肽处方。
表1.含不同功能多肽脂质纳米递药系统的表征和原代星形胶质细胞摄取的定量数据
Figure PCTCN2020128748-appb-000001
Figure PCTCN2020128748-appb-000002
Figure PCTCN2020128748-appb-000003
Figure PCTCN2020128748-appb-000004
“—”未加功能多肽。***P<0.001与未加功能多肽组存在显著性差异。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. 一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述递药系统包括脂质、递送药物和功能性穿膜肽，所述功能性穿膜肽由链接纳米载体端、富精氨酸穿膜肽、基质金属蛋白酶-9敏感肽和聚阴离子抑制肽的肽链共价连接形成。
  2. [根据细则91更正 29.01.2021]　
    根据权利要求1所述的一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述功能性穿膜肽的序列为端基乙酰化多肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGEGG中的任一所示。
  3. 根据权利要求1或2所述的一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述功能性穿膜肽与脂质的质量比为1:10-1:300，优选1:100。
  4. 根据权利要求1或2所述的一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述递送药物包括环孢素A、血管活性肽、脑啡肽、内啡肽、神经降压肽和新山地明。
  5. 根据权利要求4所述的一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述递送药物为环孢素A，且环孢素A由诱导沉淀方法制备。
  6. 根据权利要求5所述的一种靶向颅脑损伤病灶的脂质纳米递药系统，其特征在于，所述环孢素A与脂质的质量比为1:1-1:100，优选为1:4。
  7. 权利要求1所述一种靶向颅脑损伤病灶的脂质纳米递药系统在制备治疗颅脑损伤疾病药物中的应用。
  8. [根据细则91更正 29.01.2021]　
    一种修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽，其特征在于，所述功能性穿膜肽的序列为端基乙酰化多肽Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GAGA-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GG-RRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRRRRRRRRRR-PVGLIG-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EEEEEDDDDK，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-PVGLIG-EDDDDK，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GGGERGPPGPQGAARGFZGTPGL-EGGEGGEGG，Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRR-GPLGLLGC-EGGEGGEGG中的一种。
  9. 权利要求8所述修饰靶向颅脑损伤病灶的脂质纳米递药系统的功能性穿膜肽在制备靶向颅脑损伤病灶的脂质纳米递药系统中的应用。
  10. 权利要求5所述靶向颅脑损伤病灶的脂质纳米递药系统的制备方法，其特征在于，所述制备方法包括分步法或者一步法，所述分步法包括如下步骤：
    a)采用稀释诱导沉淀方法形成环孢素A的沉淀溶液；
    b)制备载环孢素A的脂质纳米递药系统；
    c)通过在上述b)制备的纳米递药系统溶液中加入所述的功能性穿膜肽，制备得到功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统；
    所述一步法是指脂质、环孢素A和功能性穿膜肽直接通过微流控芯片自组装制备得到功能性穿膜肽修饰的载环孢素A的脂质纳米递药系统。
  11. 根据权利要求10所述的靶向颅脑损伤病灶的脂质纳米递药系统的制备方法，其特征在于，步骤a)诱导沉淀方法由微流控技术实现，通过水相和含有环孢素A的醇相通过微流控芯片管道形成沉淀，其中醇相与水相体积比优选为1:1-1:100，其中优选比例为1:8。
  12. 根据权利要求10所述的靶向颅脑损伤病灶的脂质纳米递药系统的制备方法，其特征在于，步骤b)制备载环孢素A的脂质纳米递药系统采用微流控技术的连续流技术制备，通过微流控管道将含脂质相与微流控诱导沉淀方法制得的环孢素A沉淀通过自组装，制得载环孢素A的脂质纳米递药系统。
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