Bionic nano platform for targeted delivery of traditional Chinese medicine components based on biological orthogonal time sequence as well as preparation method and application of bionic nano platform

Abstract

The invention discloses a bionic nano-platform for targeted delivery of traditional Chinese medicine components based on a biological orthogonal time sequence and a preparation method and application of the bionic nano-platform. The bionic nano-platform comprises precursor bionic nanoparticles formed by entrapment of neuroprotective traditional Chinese medicine components on extracellular vesicles of mesenchymal stem cells modified by biological orthogonal groups, the biological orthogonal coordination group modified plant vesicle entraps a nerve repairing traditional Chinese medicine component to form the rear bionic nanoparticle, and the biological orthogonal group and the biological orthogonal coordination group can be efficiently connected in a living body. The platform has high endogenous property, high biological safety and high drug loading property, and can realize precise targeting and sequential delivery of drugs at ischemic focus parts. Nerve protection is started before thrombolysis and recanalization, ischemic lesion nerve repair is promoted after thrombolysis and recanalization, the nerve protection and the time sequence delivery effect of repairing traditional Chinese medicine components are achieved through biological orthogonality, and the multi-target-point, multi-layer and multi-stage treatment advantages of cerebral arterial thrombosis are exerted.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano preparations, and in particular relates to a biomimetic nano platform for targeted delivery of traditional Chinese medicine components based on bio-orthogonal timing, and a preparation method and application thereof. Background Art

[0002] Ischemic stroke is a brain tissue injury caused by a sudden reduction or interruption of blood flow due to cerebral vascular thrombosis. It accounts for about 87% of the total incidence of clinical stroke and is the leading cause of death and disability worldwide. At the onset of the disease, brain tissue in the core area of ​​thrombotic infarction rapidly necrotizes, and neuronal apoptosis and necrosis continue to occur in the ischemic penumbra of the peripheral blood flow hypoperfusion area. Clinical treatment is mainly based on intravenous infusion of alteplase (Recombinant tissue plasminogen activator, rt-PA) thrombolysis within 4.5 hours of onset. However, due to the rapid expansion of the ischemic infarction core and the strict limitation of the thrombolytic treatment time window, only a few patients can effectively receive thrombolytic therapy. Moreover, rt-PA is difficult to cope with secondary reperfusion injury after blood flow is restored. Less than 3% of patients benefit from thrombolysis. There is an urgent need for collaborative treatment strategies to improve clinical efficacy. Neuroprotection and repair therapy can inhibit the ischemic cascade reaction to reduce brain damage, thereby prolonging the recanalization treatment time window and improving ischemia-reperfusion injury. At present, clinical studies have explored the application of neuroprotection and neurorepair combined with thrombolytic therapy, such as the use of edaravone combined with butylphenol, Xingnaojing injection or Danshen injection to improve the early neurological function of patients receiving thrombolytic therapy. However, clinical combination medication is often limited to the superposition of antioxidant neuroprotective preparations, and fails to provide targeted medication strategies based on the pathological characteristics before and after thrombolysis and recanalization. The treatment model is single, and the interaction between drugs is not clear. There is a lack of accurate, coherent, and comprehensive treatment plans. Therefore, designing a multi-stage, full-chain treatment system of "neuroprotection, thrombolysis and recanalization, and neurorepair" is the key to comprehensively improving the prognosis of patients.

[0003] Compared with drugs that only target a single link or a single pathological target of the ischemic cascade, multi-component Chinese medicines with multi-target intervention effects can comprehensively regulate the ischemic injury cascade, showing the advantages of comprehensive treatment. Ischemic stroke belongs to the category of "stroke" in traditional Chinese medicine, and its core pathogenesis is "Qi deficiency and blood stasis", with Qi-invigorating and blood-activating as the main treatment method. For example, Astragalus-Salvia miltiorrhiza is a commonly used Qi-invigorating and blood-activating drug pair, which is used for cardiovascular and cerebrovascular diseases with Qi-blood deficiency and stasis. The Compendium of Materia Medica records that Salvia miltiorrhiza is often used in combination with Astragalus to enhance the function of removing blood stasis and relieving pain. Astragalus treats from the root, replenishing the middle and replenishing Qi; Salvia miltiorrhiza is good at treating blood, activating blood circulation and unblocking menstruation. Modern pharmacological basic analysis shows that the main active components of the Astragalus-Salvia miltiorrhiza drug pair include total astragaloside and total phenolic acid, which have multiple pharmacological effects such as anti-inflammatory, anti-apoptosis, neuroprotection, and vascular remodeling. However, multi-components of traditional Chinese medicine still have problems in terms of bioavailability, such as low absorption rate, poor blood-brain barrier penetration, and poor targeting. Therefore, there is an urgent need to develop a new delivery strategy to increase the accumulation of multi-components of traditional Chinese medicine in brain lesion targets, laying the foundation for the realization of a full-chain treatment system.

[0004] Extracellular vesicles (EVs) are bioactive nanoparticles produced actively by eukaryotic prokaryotic cells. They have a double-layer lipid membrane structure, transport various biomolecules between cells, and regulate intercellular communication. Extracellular vesicles transport bioactive molecules to cells of specific organs or tissues through the circulatory system, playing a key role in mediating signal transduction. Mesenchymal stem cell EVs (MEVs) have the ability to naturally home to inflammatory lesions and can autonomously migrate to the area of ​​cerebral ischemic lesions. Compared with artificial nanocarriers, MEVs have the advantages of physiological barrier permeability, easy loading of drugs, easy modification, and good biocompatibility. In recent years, plant-derived EVs have attracted widespread attention due to their rich sources and high yields. They are similar to mammalian EVs in morphology, composition, and function. Plant vesicles are derived from natural plants, have multi-component and multifunctional characteristics, are easily absorbed and utilized by organisms, can be used as biotherapeutics and drug delivery carriers, and help solve the problem of poor bioavailability of active ingredients. In addition, since plants do not have zoonotic pathogens, plant vesicles are also non-immunogenic and have high safety. However, due to the complexity of the ischemic stroke lesion environment, the efficiency of EVs in delivering drugs to the brain is low, and plant vesicles do not have cell targeting specificity, which still requires further modification and optimization. Summary of the invention

[0005] Purpose of the invention: In view of the above technical problems, the purpose of the present invention is to provide a biomimetic nano-platform based on bio-orthogonal time-sequential targeted delivery of Chinese medicine components and its preparation method and application. The biomimetic nano-platform includes precursor biomimetic nanoparticles and post-biomimetic nanoparticles, which can be combined with the characteristics of efficient and rapid brain entry through nasal administration. Before thrombolytic therapy, the inflammatory tropism of extracellular vesicles of mesenchymal stem cells is used to deliver precursor biomimetic nanoparticles to ischemic lesions to exert neuroprotective effects and quickly release bio-orthogonal groups. After thrombolytic therapy, post-biomimetic nanoparticles are administered to increase the accumulation of plant vesicle biomimetic nanoparticles in ischemic lesions through in situ bio-orthogonal reactions in tissues, and exert a neurorepair effect. In addition, the time-sequential delivery effect of neuroprotective and repair drugs in the ischemic lesion area is achieved, providing a new multi-target, multi-level, and multi-stage combined treatment strategy for the treatment of ischemic stroke.

[0006] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides a biomimetic nano-platform for targeted delivery of Chinese medicine components based on bio-orthogonal timing, the biomimetic nano-platform comprising precursor biomimetic nanoparticles formed by mesenchymal stem cell extracellular vesicles modified with bio-orthogonal groups encapsulating neuroprotective Chinese medicine components, and post-biomimetic nanoparticles formed by plant vesicles modified with bio-orthogonal ligands encapsulating neurorepairing Chinese medicine components. The bio-orthogonal groups can be directly transferred to the target tissue and can be connected with the bio-orthogonal ligands in vivo.

[0008] The traditional Chinese medicine neuroprotective component blocks the process of neuronal cell apoptosis at multiple levels by upregulating anti-apoptosis-related proteins and downregulating the levels of pro-apoptosis-related proteins, improves neuronal cell energy metabolism by protecting mitochondrial function, upregulates antioxidant enzyme expression, enhances cell survival ability, and inhibits the expansion of the core of cerebral ischemic infarction; the traditional Chinese medicine neurorepair component promotes the transformation of M1 pro-inflammatory microglia into M2 anti-inflammatory microglia, reverses the imbalance of pro-inflammatory and anti-inflammatory mediators, reduces neuroinflammatory damage, reduces neuronal cell apoptosis, repairs the damaged blood-brain barrier, and promotes angiogenesis, thereby promoting neurorepair.

[0009] As a specific embodiment, the mesenchymal stem cell extracellular vesicles are selected from one or more of the extracellular vesicles derived from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and embryonic mesenchymal stem cells; the plant vesicles are selected from one or more of the extracellular vesicles derived from ginger, ginseng, turmeric, lemon, and grapefruit.

[0010] As a specific implementation scheme, the bioorthogonal group is one or more of tetraacylated N-azidoacetylmannosamine, N-azidoacetylglucosamine, N-azidoacetylgalactosamine, and tetrazine; the bioorthogonal coordination group is one or more of dibenzocyclooctyne, bicyclo[6,1,0]nonyne, 2-cyclooctyne-acetic acid, and trans-cyclooctene.

[0011] As a specific implementation scheme, the neuroprotective Chinese medicine component is one or more of astragaloside IV, curcumin, baicalin, berberine, and salidroside; the neurorepairing Chinese medicine component is one or more of salvianolic acid B, tanshinone ⅡA, ginsenoside Rg1, rosmarinic acid, and gardenia glycoside.

[0012] In a second aspect, the present invention provides a method for preparing the biomimetic nanoplatform based on bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components, comprising the following steps:

[0013] (1) preparing mesenchymal stem cell extracellular vesicles modified with orthogonal groups;

[0014] (2) uniformly mixing the neuroprotective Chinese medicine component and the extracellular vesicles of mesenchymal stem cells obtained in step (1), ultrasonicating, incubating, and removing free drugs to obtain the precursor bionic nanoparticles;

[0015] (3) preparing plant vesicles;

[0016] (4) uniformly mixing the nerve repair Chinese medicine component and the plant vesicles obtained in step (3), ultrasonicating, and incubating to prepare bionic nanoparticles containing the nerve repair Chinese medicine component encapsulated in the plant vesicles;

[0017] (5) The biomimetic nanoparticles obtained in step (4) are surface-modified with bio-orthogonal coordination groups by covalent bonding to remove free drugs, thereby obtaining the post-positioned biomimetic nanoparticles.

[0018] As a specific embodiment, in step (1), the method for preparing orthogonal group-modified mesenchymal stem cell extracellular vesicles comprises the following steps:

[0019] Take mesenchymal stem cells, use sugar metabolism labeling method to obtain bio-orthogonal group-modified mesenchymal stem cells, collect cell culture supernatant, remove cell debris by differential centrifugation, concentrate the supernatant, add polymer precipitation, centrifuge, and obtain;

[0020] Preferably, the sugar metabolism labeling method comprises: co-incubating a polysaccharide carrying a bioorthogonal group with a mesenchymal stem cell, transferring the bioorthogonal group to the cell membrane by utilizing the sugar metabolism process, and then inducing the release of extracellular vesicles modified with the bioorthogonal group; the differential centrifugation method comprises the following: 3×10 2 Centrifuge at 2 × 10 3Centrifuge at 1 × 10 4 g centrifugation for 30-40 min; the polymer precipitation method includes the following steps: using a 100 kDa ultrafiltration tube to concentrate the supernatant, adding an extracellular vesicle separation reagent, 1.2×10 4 Centrifuge at 30-60 g for 30-60 min.

[0021] As a specific embodiment, in step (3), the method for preparing plant vesicles comprises the following steps:

[0022] Take fresh plants, squeeze the juice, remove large particles and fibers by gradient centrifugation, collect the supernatant, perform ultrafiltration, and collect the filtrate to obtain; preferably, the gradient centrifugation is performed in the following order: 2×10 3 Centrifuge at 5 × 10 3 Centrifuge at 1.5 × 10 4 g centrifugation for 55-65 min; preferably, the ultrafiltration method comprises the following: passing the supernatant through 100 kDa and 10 kDa ultrafiltration membranes in sequence.

[0023] As a specific implementation scheme, in step (2), the mass ratio of the neuroprotective Chinese medicine component to the mesenchymal stem cell extracellular vesicles is 2:(4-6) based on the protein content of the mesenchymal stem cell extracellular vesicles; the ultrasonic conditions are: the ultrasonic fragmentation time is 2s on / 5s off, 4-6min, and the ultrasonic power is 120-130W; the incubation conditions are: under 37±2°C, the incubation time is 55-65min;

[0024] As a specific implementation scheme, in step (4), the mass ratio of the nerve repair Chinese medicine component to the plant vesicles is 2:(4-6) based on the protein content therein; the ultrasonic conditions are: the ultrasonic fragmentation time is 2s on / 5s off, 4-6min, and the ultrasonic power is 120-130W; the incubation conditions are: under 37±2℃, the incubation time is 55-65min.

[0025] In the third aspect, the biomimetic nanoplatform based on bio-orthogonal time-sequence targeted delivery of Chinese medicine components of the present invention is used in the preparation of drugs for treating ischemic stroke and reperfusion injury; preferably, the drug is a nasal drug. After nasal administration, it is delivered to the brain through the olfactory nerve and trigeminal nerve pathways, with a fast onset time and simple operation, and it is easy to achieve the advantages of early ambulance emergency treatment.

[0026] In the fourth aspect, the present invention relates to the use of precursor bionic nanoparticles and post-bionic nanoparticles in combination in the preparation of drugs for the treatment of ischemic stroke and reperfusion injury, wherein the precursor bionic nanoparticles are mainly formed by mesenchymal stem cell extracellular vesicles modified with bioorthogonal groups to encapsulate neuroprotective Chinese medicine components, and the post-bionic nanoparticles are mainly formed by plant vesicles modified with bioorthogonal ligands to encapsulate neurorepair Chinese medicine components. The bioorthogonal groups can be directly transferred to the target tissue and can be connected with the bioorthogonal ligands in vivo; preferably, the drug is a nasal drug. After nasal administration, it is delivered to the brain through the olfactory nerve and trigeminal nerve pathways, with a fast onset time and simple operation, and it is easy to achieve the advantages of early ambulance emergency treatment.

[0027] When used as above, it can be dissolved by adding physiological saline, phosphate buffer, or 5% glucose solution, and administered by nasal spray or nasal inhalation. The bionic nano-platform can achieve brain-targeted delivery of Chinese medicine components across the nasal mucosal barrier, significantly improving the bioavailability of the loaded Chinese medicine components. By initiating neuroprotection before thrombolysis, the expansion of the ischemic infarction core is delayed, providing a favorable basis for thrombolysis and recanalization. After thrombolysis, by reducing secondary ischemia-reperfusion injury and promoting nerve repair in ischemic lesions, the multi-target, multi-level, and multi-stage treatment advantages of ischemic stroke are brought into play.

[0028] Bioorthogonality refers to a click chemistry reaction that can be carried out in the biological environment without interfering with normal biological processes. It has the characteristics of high specificity, rapid reactivity and biocompatibility, and can quickly form a stable chemically bonded structure at extremely low reaction substrate concentrations. It efficiently mediates the sequential delivery of drugs to achieve spatiotemporal controllability through a "two-stage" step, meets the time and space continuity of the treatment process, and conforms to the medication characteristics of the full-chain treatment system. Compared with single ligand modification, the bioorthogonal modification based on click chemistry of the present invention overcomes the "off-target" problem and provides a more efficient targeting strategy. First, the orthogonal group is integrated into the target cell surface to provide a bioorthogonal targeting site. Subsequently, the orthogonal coordination group completes the subsequent targeted delivery through in vivo bioorthogonality, thereby achieving the sequential delivery effect of different drug components in the same area and achieving an overall synergistic therapeutic effect. Therefore, the present invention realizes the sequential delivery of Chinese medicine components through a bioorthogonal strategy, and exerts a full-chain synergistic therapeutic effect. Before thrombolysis and recanalization, bionic nanoparticles of mesenchymal stem cell extracellular vesicle precursors are delivered nasally. With their inflammatory tropism, they are targeted to the ischemic lesion area and taken up by nerve cells, and the orthogonal groups are directly transferred to the nerve cells and the neuroprotective components of traditional Chinese medicine are released, avoiding the disadvantage that pre-targeted labeling requires long-term effectiveness, and achieving the dual effects of neuroprotective treatment and pre-targeted positioning. After thrombolysis and recanalization, plant vesicle post-bionic nanoparticles are administered nasally again, and delivered to the ischemic lesion with the help of in situ bioorthogonal reactions of tissues, exerting the role of nerve repair after thrombolysis and recanalization. In this way, the timed delivery of multiple components of traditional Chinese medicine in ischemic lesions is realized, achieving the purpose of full-chain treatment.

[0029] The present invention prepares orthogonal group-modified mesenchymal stem cell extracellular vesicles by sugar metabolism method, differential centrifugation method and polymer precipitation method. Compared with the traditional surface modification method, the preparation method is simple, and the modified extracellular vesicles are obtained by inducing maternal mesenchymal stem cells, and the integrity of the obtained mesenchymal stem cell extracellular vesicles is ensured. At the same time, the present invention modifies the extracellular vesicles with bioorthogonal groups, which can be directly transferred to the target site in vivo, provide bioorthogonal labeling sites, avoid the in vivo sugar metabolism labeling process, and greatly improve the efficiency of bioorthogonal targeted delivery.

[0030] The present invention prepares plant vesicles by combining ultrafiltration and differential centrifugation. Compared with the traditional ultracentrifugation method, the preparation method is simple and does not require a long ultracentrifugation process, which greatly shortens the preparation time and ensures the integrity of the obtained plant vesicles, which can be stored for a long time. In addition, the bioorthogonal coordination groups are modified on the surface of the plant vesicles by covalent modification, giving the plant vesicles stronger targeted delivery capabilities, further enhancing the effective penetration and accumulation of nerve repair drugs in the lesion site, thereby improving the delivery disadvantages of poor drug penetration and poor targeting.

[0031] The present invention selects Chinese medicine monomer compounds as therapeutic drugs, and uses ultrasonic fragmentation to induce the loading of Chinese medicine components into mesenchymal stem cell extracellular vesicles and plant vesicles, thereby improving the problem of low encapsulation rate of traditional vesicle drug loading technology.

[0032] The present invention can effectively improve the bioavailability and targeting ability of Chinese medicine components by designing a bio-orthogonally modified time-targeted delivery biomimetic nano-platform, enhance the enrichment of nerve protection and repair Chinese medicine components in ischemic lesions, and deliver drugs in a time-ordered manner at different stages of the disease to exert the therapeutic advantages of coherent treatment and comprehensive efficacy. It has the following advantages:

[0033] (1) Biosafety: The biomimetic nanoplatform based on bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components has good biosafety. It uses mesenchymal stem cells and plant-derived extracellular vesicles as delivery carriers and has the characteristics of low immunogenicity and high biosafety.

[0034] (2) Biocompatibility: Extracellular vesicles of biological origin are used to retain their endogenous characteristics, evade immune phagocytosis of macrophages in the body, and prolong the circulation time and therapeutic effect in the body.

[0035] (3) Precise targeting: The nasal delivery strategy combined with the inflammatory homing effect of mesenchymal stem cell extracellular vesicles can quickly target the brain lesion area to exert a neuroprotective effect, and directly transfer the bioorthogonal group to the target site, effectively exerting the pre-targeting positioning effect of the bioorthogonal in vivo. Plant vesicles use in situ tissue bioorthogonality to achieve targeted delivery to the brain lesion area.

[0036] (4) Sequential delivery capability: Before thrombolysis and recanalization, the precursor bionic nanoparticles initiate neuroprotection to slow down the rapid expansion of the infarct core. After thrombolysis and recanalization, the post-placed bionic nanoparticles provide neurorepair and reduce reperfusion injury. The precursor and post-placed bionic nanoparticles of traditional Chinese medicine components based on bio-orthogonal sequential targeted delivery have the characteristics of sequential delivery by acting at different stages of the disease.

[0037] (5) Full-chain holistic therapeutic effect: By using the nasal administration route combined with the in vivo bio-orthogonal "two-stage" delivery strategy, the Chinese medicine components are efficiently, quickly and sequentially delivered to the ischemic lesions at different stages, meeting the time and space continuity requirements of the treatment process. By providing neuroprotection before thrombolytic therapy and providing neurorepair after thrombolytic therapy, the full-chain treatment advantages of multiple targets, multiple levels and multiple stages are achieved.

[0038] (6) Synergistic effect of carrier therapy: As the main participant in the paracrine effect of stem cell therapy, mesenchymal stem cell extracellular vesicles contain multiple effective components and have certain neuroprotective effects; plant vesicles contain multiple plant active substances and have certain pharmacological effects. Both of them can play a synergistic role with traditional Chinese medicine components when used as drug delivery carriers.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0040] (1) The present invention adopts the sugar metabolism method, differential centrifugation method, polymer precipitation method and ultrasonic crushing method to prepare biomimetic nanoparticles of mesenchymal stem cell extracellular vesicles modified with bioorthogonal groups for delivering Chinese medicine components, and obtains the modified extracellular vesicles from the parent cell operation, which can reduce the preparation steps, ensure the integrity of the extracellular vesicles, and improve the encapsulation rate of the drug. At the same time, the bioorthogonal group can be directly transferred to the target site in vivo, providing a bioorthogonal labeling site, avoiding the sugar metabolism labeling process in vivo, and greatly improving the efficiency of bioorthogonal targeted delivery.

[0041] (2) The present invention adopts ultrafiltration combined with ultracentrifugation and ultrasonic crushing to prepare plant vesicles for delivering Chinese medicine components and post-delivery bionic nanoparticles, avoiding the long ultracentrifugation process, greatly saving time and cost, and being able to better ensure the integrity of the obtained vesicles and improve the encapsulation rate of the drug. The method is simple, low-cost, and easier to realize industrial production.

[0042] (3) The present invention selects extracellular vesicles as drug carriers, which are more diverse than liposomes, micelles, supramolecular assemblies, nanoemulsions and other dosage forms, can be loaded with hydrophilic and hydrophobic drugs, have high delivery efficiency and good stability. By utilizing its protein lipid membrane structure, it can not only deliver Chinese medicine monomer compounds, but also achieve the combined delivery of multiple components of Chinese medicine, and play a comprehensive protective role against the complex pathological process of ischemic stroke.

[0043] (4) The biomimetic nanoparticles provided by the present invention use extracellular vesicles of biological origin, which reduces the toxicity and side effects of the drug itself, improves biosafety and biocompatibility, and ensures the safety of use as a carrier.

[0044] (5) The biomimetic nanoparticles provided by the present invention achieve high drug encapsulation efficiency and drug loading, and are bio-orthogonally modified to significantly improve the biosafety and targeted delivery capability of the drug, thereby achieving precise drug delivery and target concentration at the lesion site.

[0045] (6) The bionic nanoplatform for the time-sequential delivery full-chain treatment of ischemic stroke provided by the present invention can be loaded with different treatment methods for combined treatment and active drugs in different lesion sites to achieve the effect of combined administration and time-sequential delivery. It provides a reference for the construction of a time-sequential delivery system for safe targeted treatment, multi-drug combination treatment, and simultaneous treatment of multiple lesions of ischemic stroke and other cardiovascular and cerebrovascular diseases, and has good application prospects and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1This is an investigation of the morphology and stability of N3-MEVs / AsIV and dGDNPs / SAB in Example 5 1.1, wherein: A is the transmission electron microscope image of the nanoparticles, and B is the particle size stability of the nanoparticles;

[0047] Figure 2 The surface characteristic protein expression of N3-MEVs / AsIV and dGDNPs / SAB in Example 5 1.2, wherein: A is the expression of the characteristic protein of N3-MEVs / AsIV, and B is the expression of the characteristic protein of dGDNPs / SAB;

[0048] Figure 3 It is the orthogonal group modified expression of N3-MEVs / AsIV and dGDNPs / SAB in Example 5 1.3, wherein: A is the expression of N3 on N3-MEVs / AsIV, and B is the expression of DBCO on dGDNPs / SAB;

[0049] Figure 4 This is the bio-orthogonal pre-targeted labeling investigation of N3-MEVs / AsIV in Example 5 1.4, wherein: A is the fluorescence image of N3 expression on the cell membrane, and B is the quantitative result of N3 expression on the cell membrane;

[0050] Figure 5 This is an investigation of the bioorthogonal targeted delivery of dGDNPs / SAB in Example 5 1.5, wherein: A is a fluorescence image of the bioorthogonal delivery of dGDNPs / SAB, and B is a fluorescence semi-quantitative result of the bioorthogonal delivery;

[0051] Figure 6 This is the investigation of the anti-apoptotic effect of N3-MEVs / AsIV in vitro in Example 5 1.6, wherein: A is the fluorescence image of live-dead staining of cells, and B is the fluorescence semi-quantitative result of live-dead staining;

[0052] Figure 7 This is an investigation of the effect of N3-MEVs / AsIV in improving mitochondrial function in vitro in Example 5 1.7, wherein: A is a fluorescence image of the change of mitochondrial membrane potential, and B is a fluorescence semi-quantitative result of mitochondrial membrane potential;

[0053] Figure 8 This is the investigation of the phenotypic effect of dGDNPs / SAB polarized microglia in vitro in Example 5, 1.8, wherein: A is the fluorescence image of microglial marker expression, and B is the quantitative result of marker expression;

[0054] Fig. 9 This is the investigation of the anti-neuroinflammatory effect of dGDNPs / SAB in vitro in Example 5, 1.8;

[0055] Fig.10This is the study of the delivery of N3-MEVs / AsIV in vivo in Example 5.1.9;

[0056] Fig.11 This is an investigation on the in vivo delivery of dGDNPs / SAB in Example 5, 1.9. DETAILED DESCRIPTION

[0057] The present invention is further described by the following examples. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention. The invention is further described below in conjunction with the accompanying drawings and examples:

[0058] Example 1: Extraction of N3-modified mesenchymal stem cell extracellular vesicles

[0059] Primary bone marrow mesenchymal stem cells were extracted from rat bone marrow and cultured to the 2nd-3rd generation in DMEM / F12 medium (containing FBS). The cells were cultured for 48 h in a medium containing tetraacylated N-azidoacetylmannosamine (Ac4ManNAz) to metabolize the bioorthogonal group azide (N3) to the cell membrane. The cells were cultured for another 48 h in DMEM / F12 medium without FBS to induce EVs secretion. The cell culture supernatant was collected and incubated at 4 °C for 3 × 10 2 g for 10 min to remove the cells, and 2 × 10 3 g for 20 min to remove dead cells, and 1 × 10 4 The cells were centrifuged at 3 × 10 g for 30 min to remove cell debris and the supernatant was collected. 2 g ultrafiltration for 5 min. Add EVs isolation reagent to the supernatant, mix gently, and incubate at 4 °C at 1.2 × 10 4 The pellet was resuspended in PBS and stored at 80°C for later use to obtain N3-modified MEVs (N3-MEVs).

[0060] Example 2: Preparation process of N3-MEVs (N3-MEVs / AsIV) loaded with astragaloside IV

[0061] N3-MEVs / AsIV was prepared by ultrasonic disruption. 200 μg of N3-MEVs was taken in terms of protein, 80 μg of AsIV was added, and ultrasonic disruption was performed for 5 min at 120 W and 2s on / 5s off to load AsIV into N3-MEVs. The mixture was incubated at 37°C for 1 h to close the membrane structure and remove free drugs, thereby obtaining N3-MEVs / AsIV.

[0062] Example 3: Extraction of ginger vesicles

[0063] Ginger vesicles (GDNPs) were extracted by differential centrifugation combined with ultrafiltration. Fresh ginger was peeled, juiced, and subjected to differential centrifugation (2×10 3 Centrifuge at 5 × 10 3 Centrifuge at 1.5 × 10 4 g centrifugation for 60 min) to remove large particles and fibers, and collect the supernatant. The supernatant is ultrafiltered through 100 kDa and 10 kDa filter membranes in sequence, and the filtrate is collected to obtain the product.

[0064] Example 4: Preparation of dibenzocyclooctyne (DBCO)-modified ginger vesicles (dGDNPs / SAB) loaded with salvianolic acid B (SAB)

[0065] GDBPs / SAB was prepared by ultrasonic crushing. 200 μg of GDNPs was taken in terms of protein, 80 μg of SAB was added, and ultrasonic crushing was performed for 5 min at a power of 120 W and 2s on / 5s off to load SAB into GDNPs. The mixture was incubated at 37°C for 1 h to close the membrane structure and remove free drugs, thereby obtaining GDNPs / SAB.

[0066] DBCO was modified on GDNPs by amidation reaction to construct dGDNPs / SAB. 6.0 mg EDC and 6.0 mg NHS were added to 2.0 mg DBCO-Acid to activate the -COOH group. After stirring at room temperature for 2 h, the mixture was added to GDNPs / SAB and stirred for overnight reaction. 5 The mixture was ultracentrifuged at 4000 × g for 30 min and dispersed in PBS buffer to obtain dGDNPs / SAB.

[0067] Example 5: Investigation of the properties of the precursor bionic nanoparticles N3-MEVs / AsIV and the subsequent bionic nanoparticles dGDNPs / SAB

[0068] 1.1 Morphology and stability investigation

[0069] The morphology of the nanoparticles was observed using a transmission electron microscope using N3-MEVs / AsIV and dGDNPs / SAB obtained in Example 2 and Example 4. Figure 1 As shown in A, both biomimetic nanoparticles present a spherical morphology and retain a clearly visible EVs double membrane structure. N3-MEVs / AsIV and dGDNPs / SAB were dispersed in PBS and stored at 4°C. The particle size was measured by dynamic light scattering every day to investigate their storage stability. Figure 1 As shown in B, within 5 days, N3-MEVs / AsIV and dGDNPs / SAB can maintain a constant size and have good stability.

[0070] 1.2 Expression of characteristic proteins on the surface of EVs

[0071] Take N3-MEVs / AsIV and dGDNPs / SAB obtained in Example 2 and Example 4, and use the BCA protein concentration determination method to determine the total protein content. Take equal volumes of MEVs, N3-MEVs / AsIV and GDNPs, dGDNPs / SAB solutions and RIPA lysate and mix them, react on ice for 15 minutes, take 10 μL of the reaction solution, add 10 μL of PBS buffer and mix, add 200 μL BCA working solution, shake at 37°C for 30 minutes, measure the absorbance at a wavelength of 562 nm, and calculate the protein concentration in the sample according to the standard curve.

[0072] Take 100 μg of MEVs, N3-MEVs / AsIV, GDNPs, and dGDNPs / SAB in terms of protein, add fluorescently labeled primary antibodies APC-CD63 and PE-CD81 in turn, set up corresponding isotype controls, add 500 μL of binding solution, incubate at room temperature for 20 min, pass through a 300-mesh nylon mesh, and detect protein expression by flow cytometry. Figure 2 As shown, MEVs, N3-MEVs / AsIV, GDNPs and dGDNPs / SAB all contained EVs marker proteins CD63 and CD81, indicating that N3-MEVs / AsIV and dGDNPs / SAB retained the characteristic membrane proteins on the surface of EVs.

[0073] 1.3 Expression of bioorthogonal groups

[0074] MEVs / AsIV and GDNPs / SAB that were not modified with orthogonal groups and coordination groups were prepared according to the methods of the above-mentioned Example 2 and Example 4.

[0075] Take MEVs / AsIV, N3-MEVs / AsIV, GDNPs / SAB, dGDNPs / SAB, 100 μg of protein, add fluorescent orthogonal group probes DBCO-Cy5 and N3-Cy5, incubate at room temperature for 20 min, pass through 300 mesh nylon net, and detect fluorescence expression by flow cytometry to characterize the expression of orthogonal groups on biomimetic nanoparticles. The results are shown in Figure 2. Figure 3 As shown, the fluorescence intensity of N3-MEVs / AsIV and dGDNPs / SAB was significantly higher than that of the unmodified biomimetic nanoparticles, indicating the successful modification of the bioorthogonal groups.

[0076] 1.4 Pre-targeted labeling effect of bioorthogonal reactions

[0077] Preparation of Ac4ManNAz-loaded liposomes (Lip / Ac): Lip / Ac was prepared by thin film hydration method. DMPC phospholipids and Ac4ManNAz (1:10) were dissolved in chloroform, evaporated to dryness using a rotary evaporator to form a honeycomb film, hydrated with PBS, and ultrasonically disrupted (power 120 W, 5s on / 10s off, 5min) to remove free drugs, thus obtaining Lip / Ac.

[0078] After N3-MEVs / AsIV is taken up by cells, the N3 group is transferred to the nerve cells through the membrane fusion effect, playing a pre-targeting labeling role. PC12 cells were seeded in a 12-well plate containing a confocal slide, with a cell density of 1×10 per well. 5 Cells were cultured at 37°C in 5% CO2. After the cells adhered overnight, N3-MEVs / AsIV and MEVs / AsIV were added and cultured for 4 hours. After the culture was completed, the culture medium was discarded, the cells were washed with PBS, and DBCO-Cy5 was added and cultured for 15 minutes to label the N3 group. The cells were washed with PBS and imaged by laser confocal imaging. The results are shown in Figure 4 As shown in A, after N3-MEVs / AsIV was co-incubated with PC12 cells, the fluorescence intensity of DBCO-Cy5 increased significantly, indicating that the N3 group was successfully transferred to PC12 cells and played a pre-targeting labeling role.

[0079] The cell membrane of PC12 treated with different preparations was separated using a cell membrane protein and cytoplasm protein extraction kit, and the cell membrane protein content was determined by the BCA method. 200 μL of cell membrane suspension was taken and the fluorescence intensity of DBCO-Cy5 was measured using a multifunctional microplate reader. The results are shown in Figure 4 As shown in B, after N3-MEVs / AsIV was co-incubated with PC12 cells for 4 h, the content of N3 groups on the cell membrane was significantly higher than that in the Lip / Ac and MEVs / AsIV groups, indicating that N3-MEVs / AsIV could quickly transfer N3 groups to target cells, improve the long-term onset process of glucose metabolism labeling, and improve the efficiency of pre-targeting labeling.

[0080] 1.5 In vitro orthogonal reaction investigation

[0081] Preparation of DiI fluorescently labeled biomimetic nanoparticles: Take 200 μg of GDNPs / SAB and dGDNPs / SAB in terms of protein, add 1 μL of 1 mM DiI fluorescent dye, use ultrasonic fragmentation method, power 120W, ultrasonic fragmentation for 2 min under 2s on / 5s off conditions, incubate at 37°C for 1 h to close the membrane structure, and obtain DiI fluorescently labeled biomimetic nanoparticles DiI-GDNPs / SAB and DiI-dGDNPs / SAB.

[0082] dGDNPs / SAB achieves targeted delivery effect through bioorthogonal reaction of DBCO and N3. PC12 cells were seeded in a 12-well plate containing a confocal slide with a cell density of 1×10 per well. 5 Cells were cultured at 37°C in 5% CO2. After the cells adhered overnight, N3-MEVs / AsIV were added and cultured for 4 hours. After the culture was completed, the culture medium was discarded, the cells were washed with PBS, and the DiI fluorescently labeled biomimetic nanoparticles prepared above were added and cultured for another 4 hours. The cells were imaged using a laser confocal instrument. Figure 5 As shown, compared with the unmodified biomimetic nanoparticles, the dGDNPs / SAB group showed a stronger fluorescence signal, indicating that DBCO and N3 can successfully undergo orthogonal reactions to achieve more formulation uptake.

[0083] 1.6 Investigation of neuroprotective effects

[0084] Establishment of oxygen-glucose deprivation (OGD) cell model: PC12 cells in the logarithmic growth phase were inoculated into well plates. After overnight attachment, the culture medium was discarded, and DMEM sugar-free culture medium was added. The cells were placed in an anoxic chamber and placed in a 37°C incubator for OGD treatment for 6 hours.

[0085] Calcein-AM / PI live-dead staining was used to investigate the protective effect of N3-MEVs / AsIV on damaged PC12 cells. PC12 cells were seeded in 96-well plates at a cell density of 1×10 5 Cells were added and cultured at 37°C in 5% CO2. After the cells adhered overnight, N3-MEVs / AsIV were added to perform OGD modeling. After the modeling was completed, the culture medium was discarded, the cells were washed with PBS, Calcein-AM and PI were diluted and mixed, added to the well plate, incubated for 30 minutes, observed under an inverted fluorescence microscope, and fluorescence data were analyzed using Image J software. The results are shown in Figure 6 As shown, compared with the OGD and free AsIV groups, the number of apoptotic cells in the N3-MEVs / AsIV group was significantly reduced, indicating that N3-MEVs / AsIV effectively inhibited the cell damage caused by OGD.

[0086] The JC-1 fluorescent probe labeling method was used to detect the effect of N3-MEVs / AsIV on mitochondrial membrane potential. PC12 cells were seeded in a 12-well plate containing a confocal slide at a cell density of 1×10 per well. 5cells and cultured them in 5% CO2 at 37°C. After the cells adhered overnight, N3-MEVs / AsIV were added for OGD modeling. After modeling, the culture medium was discarded, the cells were washed with PBS, and JC-1 working solution was added, mixed thoroughly, and incubated in a 37°C cell culture incubator in the dark for 20 minutes. After incubation, the supernatant was discarded and JC-1 staining buffer was added for washing. The cells were imaged using a laser confocal instrument. The results are shown in Figure 2. Figure 7 As shown in the figure, compared with the OGD and free AsIV groups, N3-MEVs / AsIV significantly reversed the decline in mitochondrial membrane potential and had a certain neuroprotective effect.

[0087] 1.7 Investigation of nerve repair effect

[0088] Establishment of lipopolysaccharide (LPS) inflammatory cell model: BV2 cells in the logarithmic growth phase were inoculated into a well plate. After overnight attachment, 1 μg / mL LPS solution was added to induce for 24 hours, and the model was completed.

[0089] The effect of dGDNPs / SAB on polarizing microglia was investigated by immunofluorescence staining of nitric oxide synthase (iNOS) and arginine-1 (Arg-1). BV2 cells were seeded in 24-well plates at a cell density of 2×10 5 cells and cultured them at 37°C in 5% CO2. After the cells adhered overnight, LPS was added to induce the inflammatory cell model. After the model was established, the culture medium was discarded and the cells were washed with PBS. dGDNPs / SAB was added for 24 hours. Fluorescent primary antibodies Alexa Fluor 594-iNOS and AlexaFluor 488-Arg-1 were used for staining. The cells were imaged using a laser confocal instrument. The results are shown in Figure 2. Figure 8 As shown in the results, compared with the LPS and free SAB groups, dGDNPs / SAB significantly enhanced the expression of Arg-1, a marker of M2 BV2 cells, and reduced the expression of iNOS, a marker of M1 cells, indicating that dGDNPs / SAB can effectively promote the polarization of microglia to the M2 phenotype and play a role in neural repair.

[0090] Enzyme-linked immunosorbent assay was used to detect the effect of dGDNPs / SAB on reducing the expression level of intracellular inflammatory factors. BV2 cells were seeded in 24-well plates at a cell density of 2×10 per well. 5cells and cultured them at 37°C in 5% CO2. After the cells adhered overnight, LPS was added to induce an inflammatory cell model. After the model was established, the culture medium was discarded and the cells were washed with PBS. dGDNPs / SAB was added for 24 hours. According to the instructions of the kit, the supernatant of the cells was collected and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10) were detected. The results are shown in Fig. 9 As shown in the results, compared with the LPS and free SAB groups, dGDNPs / SAB significantly reduced the production of pro-inflammatory cytokines and promoted the production of anti-inflammatory cytokines, indicating that dGDNPs / SAB has a good anti-inflammatory effect.

[0091] 1.8 In vivo bioorthogonal delivery investigation

[0092] Preparation of DiR fluorescently labeled biomimetic nanoparticles: Take 200 μg of N3-MEVs / AsIV, GDNPs / SAB and dGDNPs / SAB in terms of protein, add 1 μL of 1 mM DiR fluorescent dye. Use ultrasonic disruption method, power 120W, ultrasonic disruption for 2 min under 2s on / 5s off conditions, and incubate at 37°C for 1 h to close the membrane structure, thus obtaining DiR fluorescently labeled biomimetic nanoparticles DiR-N3-MEVs / AsIV, DiR-GDNPs / SAB and DiR-dGDNPs / SAB.

[0093] Construction of rat model of cerebral ischemia: Rats were anesthetized by intraperitoneal injection of sodium pentobarbital solution, and the common carotid artery, external carotid artery and internal carotid artery were separated inertly from the middle neck incision. The proximal end of the external carotid artery and common carotid artery was ligated, the common carotid artery was incised, and a thread plug was inserted and pushed from the internal carotid artery to the middle cerebral artery. When there was slight resistance, it was stopped and ligated, and the wound was sutured. The thread plug remained in the occluded state to construct a permanent middle cerebral artery occlusion model (pMCAO); after 2 hours of thread plug occlusion, the thread plug was pulled out to the internal carotid artery, and the blood supply of the middle cerebral artery was restored to prepare a transient middle cerebral artery occlusion model (tMCAO).

[0094] In the construction of pMCAO model, free DiR and DiR-N3-MEVs / AsIV were administered intranasally, and fluorescence imaging of rats was performed using a small animal in vivo imaging device at different time periods. Fig.10 As shown, the fluorescence intensity of DiR-N3-MEVs / AsIV in the brain was significantly higher than that of free DiR, indicating that N3-MEVs / AsIV can be rapidly delivered to the brain by virtue of its inflammatory tropism.

[0095] In the construction of tMCAO model, N3-MEVs were first administered intranasally to achieve bioorthogonal pre-labeling effect. Then DiR-GDNPs / SAB and DiR-dGDNPs / SAB were administered intranasally, and fluorescence imaging of rats was performed using a small animal in vivo imaging device at different time periods. Fig.11 As shown, the fluorescence intensity of DiR-dGDNPs / SAB in the brain was significantly higher than that of the unmodified biomimetic nanoparticles DiR-GDNPs / SAB, indicating that dGDNPs / SAB achieved targeted delivery through in vivo bioorthogonal reaction.

Claims

1. A biomimetic nanoplatform based on bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components, characterized in that: The bionic nano-platform includes precursor bionic nanoparticles formed by mesenchymal stem cell extracellular vesicles modified with bioorthogonal groups encapsulating nerve protection Chinese medicine components, and post-bionic nanoparticles formed by plant vesicles modified with bioorthogonal ligands encapsulating nerve repair Chinese medicine components. The bioorthogonal groups and bioorthogonal ligands can be connected in vivo.

2. The biomimetic nanoplatform based on bioorthogonal time-sequential targeted delivery of traditional Chinese medicine components according to claim 1, characterized in that: The mesenchymal stem cell extracellular vesicles are selected from one or more of the extracellular vesicles derived from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and embryonic mesenchymal stem cells; the plant vesicles are selected from one or more of the extracellular vesicles derived from ginger, ginseng, turmeric, lemon, and grapefruit.

3. The biomimetic nanoplatform based on bioorthogonal time-sequential targeted delivery of traditional Chinese medicine components according to claim 1, characterized in that: The bioorthogonal group is one or more of tetraacylated N-azidoacetylmannosamine, N-azidoacetylglucosamine, N-azidoacetylgalactosamine, and tetrazine; the bioorthogonal coordination group is one or more of dibenzocyclooctyne, bicyclo[6,1,0]nonyne, 2-cyclooctyne-acetic acid, and trans-cyclooctene.

4. The biomimetic nanoplatform based on bioorthogonal time-sequential targeted delivery of traditional Chinese medicine components according to claim 1, characterized in that: The neuroprotective Chinese medicinal component is one or more of astragaloside IV, curcumin, baicalin, berberine, and salidroside; the neurorepairing Chinese medicinal component is one or more of salvianolic acid B, tanshinone ⅡA, ginsenoside Rg1, rosmarinic acid, and gardenia glycoside.

5. The method for preparing a biomimetic nanoplatform for bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) preparing mesenchymal stem cell extracellular vesicles modified with orthogonal groups; (2) uniformly mixing the neuroprotective Chinese medicine component and the extracellular vesicles of mesenchymal stem cells obtained in step (1), ultrasonicating, incubating, and removing free drugs to obtain the precursor bionic nanoparticles; (3) preparing plant vesicles; (4) uniformly mixing the nerve repair Chinese medicine component and the plant vesicles obtained in step (3), ultrasonicating, and incubating to prepare bionic nanoparticles containing the nerve repair Chinese medicine component encapsulated in the plant vesicles; (5) The biomimetic nanoparticles obtained in step (4) are modified with bio-orthogonal coordination groups by covalent bonding, and free drugs are removed to obtain the post-positioned biomimetic nanoparticles.

6. The method for preparing a biomimetic nano-platform for bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components according to claim 5, characterized in that: In step (1), the method for preparing orthogonal group-modified mesenchymal stem cell extracellular vesicles comprises the following steps: Take mesenchymal stem cells, use sugar metabolism labeling method to obtain bio-orthogonal group-modified mesenchymal stem cells, collect cell culture supernatant, remove cell debris by differential centrifugation, concentrate the supernatant, add polymer precipitation, centrifuge, and obtain; Preferably, the sugar metabolism labeling method comprises: co-incubating a polysaccharide carrying a bioorthogonal group with a mesenchymal stem cell, transferring the bioorthogonal group to the cell membrane by utilizing the sugar metabolism process, and then inducing the release of extracellular vesicles modified with the bioorthogonal group; the differential centrifugation method comprises the following: 3×10 2 Centrifuge at 2 × 10 3 Centrifuge at 1 × 10 4 g centrifugation for 30-40 min; the polymer precipitation method includes the following steps: using a 100 kDa ultrafiltration tube to concentrate the supernatant, adding an extracellular vesicle separation reagent, 1.2×10 4 Centrifuge at 30-60 g for 30-60 min.

7. The method for preparing a biomimetic nano-platform for bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components according to claim 5, characterized in that: In step (3), the method for preparing plant vesicles comprises the following steps: Take fresh plants, squeeze the juice, remove large particles and fibers by gradient centrifugation, collect the supernatant, perform ultrafiltration, and collect the filtrate to obtain; preferably, the gradient centrifugation is performed in the following order: 2×10 3 Centrifuge at 5 × 10 3 Centrifuge at 1.5 × 10 4 g centrifugation for 55-65 min; preferably, the ultrafiltration method comprises the following: passing the supernatant through 100 kDa and 10 kDa ultrafiltration membranes in sequence.

8. According to the method for preparing a biomimetic nanoplatform for bio-orthogonal sequential targeted delivery of Chinese medicine components in claim 5, in step (2), the mass ratio of the mesenchymal stem cell extracellular vesicles is 2:(4-6) based on the protein content therein; the ultrasonic conditions are: the ultrasonic fragmentation time is 2s on / 5s off, 4-6min, and the ultrasonic power is 120-130W; the incubation conditions are: under 37±2°C, the incubation time is 55-65min; In step (4), the mass ratio of the nerve repair Chinese medicine component to the plant vesicles is 2:(4-6) based on the protein content of the plant vesicles; the ultrasonic conditions are: the ultrasonic fragmentation time is 2s on / 5s off, 4-6min, and the ultrasonic power is 120-130W; the incubation conditions are: at 37±2°C, the incubation time is 55-65min.

9. Use of the biomimetic nanoplatform based on bio-orthogonal time-sequential targeted delivery of traditional Chinese medicine components as described in any one of claims 1 to 4 in the preparation of drugs for treating ischemic stroke and reperfusion injury; preferably, the drug is a drug for nasal administration.

10. Use of precursor bionic nanoparticles and post-bionic nanoparticles in combination in the preparation of drugs for treating ischemic stroke and reperfusion injury, characterized in that: The precursor bionic nanoparticles are mainly formed by mesenchymal stem cell extracellular vesicles modified with bioorthogonal groups encapsulating neuroprotective Chinese medicinal components, and the post-bionic nanoparticles are mainly formed by plant vesicles modified with bioorthogonal ligands encapsulating neurorepairing Chinese medicinal components, and the bioorthogonal groups and bioorthogonal ligands can be connected in vivo; preferably, the drug is a drug for nasal administration.

Images