CN115212188A

CN115212188A - Ischemic brain region targeted nanowire mitochondria and preparation method and application thereof

Info

Publication number: CN115212188A
Application number: CN202210906755.2A
Authority: CN
Inventors: 高巍; 宋珂珂; 王强; 闫超颖; 王妮莎; 夏季; 张闪闪
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2022-07-29
Filing date: 2022-07-29
Publication date: 2022-10-21
Anticipated expiration: 2042-07-29
Also published as: CN115212188B

Abstract

The invention belongs to the technical field of medicines, and relates to an ischemic brain region targeted nanowire granule, and a preparation method and application thereof. The preparation of the targeted nanowire mitochondria in the ischemic brain region comprises the following steps: (1) preparing SB-amylose; (2) preparation of TPP-SB-amylose; (3) preparing TPP-SB-amylose-Lf-SHp; and (4) preparing the ischemic brain region targeted nanowire mitochondria. The invention selects nano-scale mitochondria, takes amylose as a packaging material framework, connects 3-dimethyl (chloropropyl) ammonium propanesulfonate to improve the anti-phagocytosis capability of the mitochondria, efficiently carries the mitochondria to a stroke brain area in a progressive way by 'Lf receptor mediated BBB targeting' and 'SHp receptor mediated ischemic brain area targeting' and then takes the nanoparticles as a functional kernel to treat IS, thereby realizing minimally invasive transplantation and efficient and rapid targeted treatment of the damaged brain area.

Description

Ischemic brain region targeted nanowire mitochondria and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to an ischemic brain region targeted nanowire granule, and a preparation method and application thereof.

Background

Cerebral apoplexy is one of dangerous diseases with high fatality rate and high disability rate, and cerebral ischemia and hypoxia are caused by rupture bleeding or occlusion of blood vessels supplying cerebral blood, and are clinically classified into hemorrhagic disease and ischemic disease. 69.6 to 70.8 percent of cerebral apoplexy in China belongs to Ischemic Stroke (IS), the 1-year fatality rate IS 14.4 to 15.4 percent, and the disability rate IS 33.4 to 33.8 percent. At present, specific IS treatment mainly aims at improving cerebral blood circulation through cerebrovascular recanalization and assists in neuroprotection, but recanalization treatment has the problems of short treatment time window, more contraindications, ischemic reperfusion injury, cerebral hemorrhage complications and the like, and the curative effect of a neuroprotective medicament IS not fully proved.

The pathological mechanism of IS IS complex and staggered, mitochondria are organelles with various functions and forms, participate in the pathological mechanisms of IS energy metabolism, oxidative stress, inflammation, fusion, division, autophagy, apoptosis, necrosis and the like, and the early mitochondria of IS can promote cell repair through inflammation, autophagy, mitochondrial generation, fusion, division, intercellular transport, induction of endogenous protection factors and pathways and the like; in the IS late stage mitochondria, intracellular lipid, protein and nucleic acid are damaged and an apoptosis signal channel IS activated by reducing ATP generation, sodium calcium overload, active oxygen free radical increase, opening of a mitochondrial membrane permeability transition pore, inflammation, autophagy and the like, so that apoptosis and necrosis are caused. Therefore, mitochondria play a key role in the IS damage and repair process, such as early recovery of mitochondrial function and quantity, inhibition of cascade damage response and promotion of nerve repair, so the mitochondria can become an important target for treating ischemic stroke, and the study on how to early repair mitochondria has important significance for effectively treating IS.

In order to repair part of functions of mitochondria in early stage, various targeted drugs for treating mitochondria are developed at home and abroad, but the mitochondrial targeted drugs with different functions have different problems: (1) the mitochondria oxidative stress resistant medicine has too short circulation half-life, poor permeability of Blood Brain Barrier (BBB), and the single anti-oxidation treatment can also cause contradiction between reduction and oxidation, and excessive use can cause oxidative stress of cells to cause cell death. (2) Targeted repair of mitochondrial permeability provides only short-term protection, and intervention measures no longer have neuroprotective effects at a later stage. (3) Inhibiting downstream apoptosis effector, and only has short-term protection effect after ischemia; the regulation of upstream apoptosis signals such as ASK1/JNK and the like can realize the brain protection effect of ischemia for a long time, but the JNK is wide in tissue distribution, multiple in physiological characteristics and multiple in interaction channels, is not suitable for non-specifically inhibiting all subtypes, and is still difficult to avoid participating in other diseases even if only JNK3 is inhibited. Therefore, the mitochondrion-targeted drug can only repair the structure and function of the mitochondrion part by means of a single molecule, a single strategy of a pathway, a function or a form, and IS difficult to more effectively improve the neural injury of the IS. Correspondingly, the whole mitochondria IS transplanted to an ischemic brain region by adopting a 'whole strategy' of transplanting the whole mitochondria, so that the damaged mitochondria structure and function can be more comprehensively replaced and repaired, and the mitochondria transplantation IS expected to become an effective strategy for treating IS.

Mitochondrial transmission exists in the natural organism, and intercellular transfer can be realized in the mode of nano-orbits or microbubbles among neurons, mesenchymal stem cells, astrocytes, microglia, endothelial cells and other similar or dissimilar cells. In neurons, mitochondria concentrate on presynaptic nerve endings and, due to limitations in long-distance transport, post-ischemic mitochondria often fail to rapidly replenish damaged neurons. The artificially transplanted mitochondria can enter the neurons, ATP and mitochondrial DNA are provided for the damaged neurons, the anti-oxidation capability of the cells is enhanced, the ROS release is reduced, the calcium overload is relieved, meanwhile, signals such as Src/Syk and the like mediated by the integrin can participate in the endocytosis of the neurons mitochondria, the secretion of proinflammatory cytokines is triggered, the anti-apoptosis signal is up-regulated, and the stroke damage is relieved. Therefore, artificial transplantation of mitochondria may provide a new approach for IS treatment.

At present, the mitochondrial transplantation technology mainly adopts a co-culture method, a microinjection method, a liposome transfection method, a small peptide labeling method and the like, and is used for mitochondrial gene defects, myocardial ischemia, parkinson, non-alcoholic fatty liver, tumors, aging and the like. The method IS suitable for in vitro and small-amount cell mitochondrial transplantation, has the defects of low internalization rate, poor curative effect, large trauma of administration mode and the like, IS not suitable for treating acute IS, and needs to realize transformation by means of a nanotechnology.

The intravenous injection nanometer preparation enters blood circulation, the particle size is more than 500nm and is easy to be cleared by liver, the particle size is more than 200nm and is easy to be phagocytized by mononuclear phagocyte system, the particle size is less than 200nm and has the functions of enhancing penetration and prolonging circulation time, the particle size is less than 50nm and is easy to be cleared by bile, the particle size is less than 20nm and is easy to be cleared by kidney, so the ideal particle size range of the intravenous injection nanometer preparation is 50-200nm. Meanwhile, the biggest difficulty in treating IS with drugs IS that the drugs are difficult to pass through the BBB. In order to repair damaged brain tissues early, the mitochondrial preparation can be modified to have the particle size of less than 200nm and positive charges by means of BBB (brain-based b) specific receptor-mediated endocytosis through BBB by referring to macromolecular drugs, and is helpful for passing through BBB by means of BBB specific receptor-mediated active endocytosis transfer. Therefore, mitochondria with a particle size <200nm are the basis for phagocytosis by the anti-mononuclear phagocyte system and passage through the blood-brain barrier. The diameter of the natural mitochondria IS about 0.5-1 μm, the length IS 1.5-3 μm, the natural mitochondria IS easy to be cleared by the liver by direct intravenous injection, and IS easy to be phagocytized by a mononuclear phagocyte system, and the opportunity of treating IS IS lost. However, reducing the size of mitochondria by crushing, it causes damage to mitochondria, and it cannot improve cell viability and also causes toxicity.

Inflammation is induced by direct intravenous injection of mitochondria, which are phagocytosed by neutrophils, and the opportunity of treating cerebral apoplexy is lost. The brain trauma itself also prompts the organism to generate a large amount of mitochondrial microvesicles to be released into the blood, the envelope structure of the brain trauma is rich in cholesterol, phospholipid, functional receptors and the like, the brain trauma interacts with a plurality of ligand receptors, starts the external path of blood coagulation, promotes inflammatory reaction, and participates in early repair and late brain tissue injury. Therefore, there is a need to modify the mitochondrial surface structure to reduce immune phagocytosis and brain tissue damage. At the same time, the number of mitochondria-targeted "rivets" should be minimized to reduce the impact on the structure of the inner and outer membranes of mitochondria.

Based on the above studies, mitochondria need to rapidly pass through BBB to reach ischemic areas in order to improve drug efficacy, and therefore anti-phagocytosis, targeted surface modification of ischemic brain areas are needed.

Disclosure of Invention

In view of the above technical problems, the present invention provides the following technical solutions:

the invention provides a preparation method of targeted nanowire granules in an ischemic brain region, which comprises the following steps:

grafting 3-dimethyl (chloropropyl) ammonium propanesulfonate on amylose for hydrophilic modification to obtain SB-amylose;

mixing the SB-amylose with carboxyl modified triphenylphosphine (TPP-COOH) for condensation reaction to obtain TPP-SB-amylose;

the TPP-SB-amylose is subjected to hydroformylation treatment, the obtained TPP-SB-hydroformylation amylose is connected with amino groups on the homing peptide and lactoferrin to obtain TPP-SB-hydroformylation amylose-Lf-SHp, and the TPP-SB-hydroformylation amylose-Lf-SHp is subjected to reduction reaction to obtain TPP-SB-amylose-Lf-SHp;

and (3) extracting nano mitochondria from isolated blood, mixing the TPP-SB-amylose-Lf-SHp with the nano mitochondria, and self-assembling to obtain the ischemic brain region targeted nano mitochondria.

Preferably, the mass ratio of the 3-dimethyl (chloropropyl) ammonium propanesulfonate to the amylose is 1: 30-60;

the mass ratio of the SB-amylose to the carboxyl modified tetraphenylpyrazine is 50-100: 1;

the mass ratio of the TPP-SB-hydroformylation amylose to the homing peptide to the lactoferrin is 10-20: 1:1;

the mol ratio of the TPP-SB-amylose-Lf-SHp to the nano mitochondria is 1-20: 1.

Preferably, the hydrophilic modification is continuous stirring at 60 ℃ for 6h;

the condensation reaction is to mix SB-amylose and carboxyl modified triphenylphosphine, add a condensing agent, react for 12 hours in an inert atmosphere and in a dark condition, carry out alcohol precipitation on reactants, and collect precipitates to obtain TPP-SB-amylose;

the hydroformylation treatment is to stir for 2 to 4 hours under the conditions of oxidant and 0 ℃ in a dark place to obtain TPP-SB-hydroformylation amylose;

the TPP-SB-hydroformylation amylose is connected with the amino groups on the homing peptide and the lactoferrin by adding the lactoferrin and the homing peptide into a TPP-SB-hydroformylation amylose solution and incubating for 3-5h at 4 ℃.

Preferably, the extraction of the nano mitochondria is to extract the mitochondria by taking isolated blood and carrying out multiple differential centrifugation, and then sequentially carrying out fractional filtration by using cell filters with the particle size of 0.45 mu m and 0.22 mu m to obtain the nano mitochondria with the particle size of less than 200nm.

Preferably, the specific operation steps of the self-assembly are: mixing the nano mitochondria and TPP-SB-amylose-Lf-SHp, magnetically stirring for 30-60min at 4 ℃, and centrifugally separating by using a mitochondria buffer solution to obtain target nano mitochondria of the ischemic brain area;

the mitochondrial buffer solution is prepared from the following raw materials in parts by weight: 0.5 part of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, 2 parts of mannitol, 70 parts of sucrose, 5 parts of 3- (N-morphinone) propanesulfonic acid, 1000 parts of water, and the pH is 7.4.

The invention also provides the ischemic brain region targeted nanowire mitochondria prepared by any one of the methods.

The invention also provides application of the ischemic brain region targeted nano mitochondria in preparation of ischemic brain region targeted drugs.

The invention also provides application of the ischemic brain region targeted nanowire granules in preparing a brain repair medicine.

The invention also provides a medicine containing the targeted nano mitochondria of the ischemic brain region.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention uses amylose as a packaging material framework, and connects 3-dimethyl (chloropropyl) ammonium propanesulfonate to obtain SB-amylose, wherein the hydroxyl of the SB-amylose is connected with the carboxyl condensed covalent bond of a nanometer mitochondrial target TPP-COOH to form the TPP-amylose. TPP is hydrophobic positive charge, and is fixed on the inner mitochondrial membrane by virtue of charge adsorption and hydrophobic acting force. TPP-amylose can be connected with SB, lf and SHp amino after hydroformylation to form a soft fish bone type TPP-SB-amylose-Lf-SHp packing material, namely the packing material is shaped like fish bone after being spread and spread after modification, and is wrapped on the surface of the nano-particle body through self-assembly of nanotechnology; the TPP-SB-amylose-Lf-SHp packing material selects amylose as an intermediate skeleton, covers the surface of mitochondria, has small particle size increase, reduces the number of fish bones inserted into the mitochondria, and reduces the influence on the functions of the mitochondria. The 'fishbone' on the side of the mitochondria is positive-charged and hydrophobic mitochondria target head TPP and is connected with the mitochondria inner membrane; the fish bone on the opposite side of the mitochondria is hydrophilic SB, lf and SHp which are externally spread in a hydrophilic environment to improve the hydrophilicity of the carrier; SB has the stealth recognition function of an immune system, thereby enhancing the anti-phagocytosis performance of IS-nMito and prolonging the peripheral circulation time; after the Lf receptor mediates IS-nMito pass through BBB, SHp relay mediates targeting to a cerebral ischemic region; repair functions are exerted by the IS-nMito internal mitochondria.

2. The invention selects the platelets of the isolated blood as the source of mitochondria, the platelets are the conventional project of blood donation, and the invention has the advantages of mature extraction process and easily obtained source; platelets are small (2-4 mm) disk-shaped nuclear cell fragments, without nuclei, but rich in mitochondria, with about 4 mitochondria (50-500 nm) in 1 platelet,<the mitochondria with 200nm is about 20%, and the normal content of platelets in venous blood of healthy people is (100-300) × 109/L<The 200nm mitochondria content is about (25-75) × 10 ⁹ and/L, the platelets can provide a rich source for the nano mitochondria.

3. The invention selects nano mitochondria, takes amylose as a packaging material framework, connects 3-dimethyl (chloropropyl) ammonium propanesulfonate to improve the anti-phagocytosis capability of the mitochondria, carries the mitochondria to a stroke brain area in a high-efficiency progressive manner by 'Lf receptor mediated BBB targeting' and 'SHp receptor mediated ischemic brain area targeting' and then takes the nanoparticles as a functional core to treat IS, and integrates 'primary antibody, secondary target and tertiary modification' design strategies, thereby realizing the design expectation of minimally invasive transplantation and high-efficiency rapid targeted treatment of the damaged brain area.

Drawings

FIG. 1 is an infrared spectrum of a synthesized SB-amylose;

FIG. 2 is an infrared spectrum of synthesized TPP-SB-amylose;

FIG. 3 is an infrared spectrum of synthesized TPP-SB-amylose-Lf-SHp;

FIG. 4 IS a graph of the morphology of natural mitochondria and IS-nSiO, particle size distribution; A. TEM images of native mitochondria (200 nm on scale); B. (ii) natural mitochondrial marvens hydrated particle size distribution; C. IS-nMito TEM (scale 100 nm); D. IS-nMo Malvern hydration particle size distribution;

FIG. 5 is near infrared tagged small animal imaging;

FIG. 6 is a TCC stained brain slice.

Detailed Description

The present invention will be further described in detail with reference to specific embodiments. The equipment and reagents used in the examples and test examples were commercially available without specific reference. The embodiments described in these examples are intended only to illustrate the invention and should not be construed as limiting the invention.

For a better understanding of the invention, without limiting its scope, all numbers expressing quantities, times, percentages, and other numerical values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired results sought to be obtained.

The invention provides a preparation method of a targeted nanowire granule in an ischemic brain region, which comprises the following steps:

preparation of SB-amylose: grafting 3-dimethyl (chloropropyl) ammonium propanesulfonate on amylose for hydrophilic modification to obtain SB-amylose;

preparation of TPP-SB-amylose: mixing the SB-amylose with carboxyl modified triphenylphosphine for condensation reaction to obtain TPP-SB-amylose;

preparation of TPP-SB-amylose-Lf-SHp: the TPP-SB-amylose is subjected to hydroformylation treatment, the obtained TPP-SB-hydroformylation amylose is connected with amino groups on the homing peptide and lactoferrin to obtain TPP-SB-hydroformylation amylose-Lf-SHp, and the TPP-SB-hydroformylation amylose-Lf-SHp is reduced under the action of a reducing agent to obtain TPP-SB-amylose-Lf-SHp;

preparing target nano mitochondria of ischemic brain region: and (3) taking isolated blood, performing multiple differential centrifugal separation to extract the nano mitochondria, mixing the TPP-SB-amylose-Lf-SHp with the nano mitochondria, and performing self-assembly to obtain the ischemic brain region targeted nano mitochondria.

The following description will be given with reference to specific examples.

Example 1

An ischemic brain region targeted nano mitochondria (IS-nSiO) IS prepared according to the following method:

(1) Preparation of SB-amylose:

12.15g of 1-chloro-3-dimethylaminopropane and 18.3g of 1, 3-propanesultone were dissolved in 100mL of 1, 2-dichloroethane and reacted at 70 ℃ for 6 hours. After the reaction is finished, the precipitate is collected and washed by 1, 2-dichloroethane to obtain the etherifying agent 3-dimethyl (chloropropyl) ammonium propanesulfonate (DCAPS). 2.78g amylose was dissolved in 10mL NaOH (11%, w/v) solution, 10mL DCAPS solution at 4.5mg/mL was added and stirring was continued at 60 ℃ for 6h. And after the reaction is finished, precipitating and separating out by using absolute ethyl alcohol, filtering, washing, drying and grinding to prepare the SB-amylose.

(2) Preparation of TPP-SB-amylose:

100mg of SB-amylose is dissolved in 20mL of Dimethylformamide (DMF), 400mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), 140mg of 1-Hydroxybenzotriazole (HOBT) and 1mg of TPP-COOH are added, the mixture is fully dissolved under magnetic stirring, the mixture is reacted overnight at room temperature in a nitrogen protection and dark place, and after the reaction is finished, the TPP-SB-amylose is prepared by precipitation by absolute ethyl alcohol, suction filtration, washing, drying and grinding.

(3) Preparation of TPP-SB-amylose-Lf-SHp:

dialyzing 100mL of TPP-SB-amylose solution with the mass fraction of 1% in sodium acetate buffer solution for 2h, sucking out the solution, putting the solution into a conical flask, and adding 0.1mol/L NaIO ₄ 100-500mL, and magnetically stirring at 0 ℃ for 2-4h in a dark place; the solution is put into a dialysis bag and dialyzed in 0.15mol/L NaCl of large volume for 2 to 3 hours, and then 20mmol/L NaHCO is put into the bag ₃ Middle dialysis is carried out for 2-3h. Adding 100mL of Lf and SHp solution with the concentration of 0.1mg/mL into 100mL of 1mg/mL hydroformylation TPP-SB-amylose solution, incubating at 4 deg.C for 5h, adding 10mL of 15.9mmol/L NaBH ₄ The solution was reacted at 4 ℃ for 24 hours, and then dialyzed against Phosphate Buffered Saline (PBS) to remove small-molecule impurities. Based on the fact that the water solubility of TPP-SB-amylose-Lf-SHp is far better than that of TPP-SB-amylose solution, in order to facilitate later-stage product expansion, a TPP-SB-amylose feeding supersaturation strategy is adopted, the main components of the product are the TPP-SB-amylose solution and the TPP-SB-amylose-Lf-SHp, the product is dispersed in an aqueous solution, the TPP-SB-amylose-Lf-SHp is dissolved in water, the TPP-SB-amylose in the product is removed through centrifugation, the TPP-SB-amylose-Lf-SHp is separated out through adding absolute ethyl alcohol into supernate, the TPP-SB-amylose-Lf-SHp is separated out through centrifugation, and the supernate is freeze-dried for later use.

(4) Preparation of IS-nMito:

and (3) separating nano platelet mitochondria: according to the specification of a platelet mitochondria kit, taking 200g of blood of an isolated healthy donor, centrifuging at a low speed for 10min, taking supernatant, removing sediment, centrifuging at 3000g for 20min, removing supernatant, taking sediment, adding a mitochondria extracting solution, and extracting mitochondria by multiple differential centrifugation. The nano mitochondria with the particle size of less than 200nm can be obtained by fractional filtration by adopting a 0.45 mu m and 0.22 mu m cell filter.

Mixing the nano mitochondria and TPP-SB-amylose-Lf-SHp in a molar ratio of 1:1, and magnetically stirring for 30min at 4 ℃. Preparing a mitochondrial buffer solution: 0.5X 10 ^-3 mol/L ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), 2X 10 ^-3 mol/L mannitol, 70X 10 ^-3 mol/L sucrose, 5X 10 ^-3 mol/L3- (N-morphinone) propanesulfonic acid, adjusting pH to 7.4. And (3) centrifugally separating the nano brain targeting mitochondria by using a mitochondrial buffer solution to remove excessive TPP-SB-amylose-Lf-SHp.

And (3) verifying and synthesizing TPP-SB-amylose-Lf-SHp by infrared spectroscopy. As shown in FIG. 1, the infrared spectrum of SB-amylose is 1197cm ^-1 And 1469cm ^-1 The characteristic peaks correspond to the asymmetric shock absorption of S = O and the N-C stretching shock absorption, indicating successful grafting of DCAPS onto amylose.

FIG. 2 shows the IR spectrum of 1697cm for TPP-SB-amylose ^-1 The characteristic peak corresponds to the characteristic absorption peak of-C = O-and is accompanied by the appearance of the characteristic absorption peak of the benzene ring, indicating that TPP has been successfully synthesized onto SB-amylose.

FIG. 3 shows the IR spectrum of TPP-SB-amylose-Lf-SHp for partial lactoferrin (1394 cm) ^-1 Primary amide C-N characteristic absorption) and stroke-homing peptide (840 cm) ^-1 N-H flexural vibration), indicating that both polypeptides have grafted onto amylose.

Example 2

The preparation method of the ischemic brain region targeted nano mitochondria is different from that of the example 1 in the preparation process of SB-amylose, and specifically comprises the following steps: 2.78g amylose was dissolved in 10mL NaOH (11%, w/v) solution, 10mL DCAPS solution at 10mg/mL was added and stirring was continued at 60 ℃ for 6h.

Example 3

The preparation method of the ischemic brain region targeted nano mitochondria is different from that of the embodiment 1 in the preparation process of TPP-SB-amylose, and specifically comprises the following steps: 100mg of SB-amylose was dissolved in 20mL of Dimethylformamide (DMF), and 400mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), 140mg of 1-Hydroxybenzotriazole (HOBT) and 2mg of TPP-COOH were added thereto, and the mixture was sufficiently dissolved with magnetic stirring and reacted at room temperature under nitrogen protection and in the dark overnight.

Example 4

The preparation method of the ischemic brain region targeted nano mitochondria is different from that of the embodiment 1 in the preparation process of TPP-SB-amylose, and specifically comprises the following steps: 80mg of SB-amylose was dissolved in 20mL of Dimethylformamide (DMF), and 400mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), 140mg of 1-Hydroxybenzotriazole (HOBT) and 1mg of TPP-COOH were added and sufficiently dissolved under magnetic stirring, and reacted overnight at room temperature in the dark under nitrogen protection.

Example 5

The preparation method of the ischemic brain region targeted nano mitochondria is different from that of the embodiment 1 in the preparation process of TPP-SB-amylose-Lf-SHp, and specifically comprises the following steps: adding 500mL of Lf and SHp solution with the concentration of 0.1mg/mL into 100mL of 10mg/mL hydroformylation TPP-SB-amylose solution, incubating at 4 ℃ for 3h, adding 50mL of 15.9mmol/L NaBH ₄ The solution was reacted at 4 ℃ for 24 hours.

Example 6

The preparation method of the ischemic brain region targeted nano mitochondria IS different from that of the example 1 in the preparation process of IS-nMito, and specifically comprises the following steps: mixing the nano mitochondria and TPP-SB-amylose-Lf-SHp in a molar ratio of 1: 20, and magnetically stirring for 60min at 4 ℃.

Example 7

The preparation method of the ischemic brain region targeted nano mitochondria IS different from that of the embodiment 1 in the preparation process of IS-nMito, and specifically comprises the following steps: mixing the nano mitochondria and TPP-SB-amylose-Lf-SHp in a molar ratio of 1: 10, and magnetically stirring for 60min at 4 ℃.

Since the performance of the ischemic brain region-targeting nanowires prepared in examples 1 to 7 is substantially the same, the following description will be given only by taking the ischemic brain region-targeting nanowires prepared in example 1 as an example.

Experimental example 1

IS-nMito structural analysis

1. Micro-morphology

The IS-nMito and natural platelet mitochondria prepared in example 1 were collected by centrifugation, fixed in a fixative, and sectioned, and changes in morphology, size and mitochondrial structure were observed by Transmission Electron Microscopy (TEM). And (3) placing the IS-nMito, the unmodified nano mitochondria and the natural platelet mitochondria on a silicon wafer, air-drying, scanning the surface morphology by adopting an atomic force microscope, and observing the morphology, the size and the surface structure of the mitochondria.

As shown in FIG. 4A, the natural mitochondrial suspension was dropped on a copper mesh, and observed by TEM, the morphology of the natural mitochondria was heterogeneous and showed rod-like shapes with different lengths, 1-2 μm in length and 0.4-0.6 μm in width.

The IS-nMito suspension was dropped on a copper mesh, and observed by TEM, the IS-nMito form a uniform spherical structure with a particle size of about 100nm, and TPP-SB-amylose-Lf-SHp coating was found to wrap around the surface of mitochondria (FIG. 4C).

2. Particle size and surface charge distribution

The particle size of the natural mitochondria, IS-nSiO preparation, was measured using a Malvern particle sizer.

As shown in FIG. 4B, the average Malvern hydrated particle size of native mitochondria was 742nm, PDI was 0.422, the first peak was 1237nm, and the second peak was 360nm.

The Malvern hydrated particle size of the IS-nMito preparation IS 183nm (figure 4D), PDI IS 0.166, and the peak value IS 156nm, which accords with the design of IS-nMito nano apparent morphology of the invention.

Experimental example 2

Targeting of IS-nMito formulations

1. Establishment of in-vivo ischemic stroke model-MCAO model

The weight of a male nude mouse is 18-20 g, the nude mouse is anesthetized by isoflurane, the middle cerebral artery of the nude mouse is blocked by adopting a wire-tying method, an ischemic stroke model is established, and the cerebral apoplexy model is perfused for 24 hours after 3 hours of blocking. Drilling a small hole at a position which is 1.5mm behind bregma of a skull of a nude mouse and is vertical to the right side of a median suture of the skull, and is a cerebral cortex ischemic central area after the middle cerebral artery is blocked, inserting a Doppler blood flow meter (LDF) optical fiber probe (with the diameter of 0.5 mm) to a cerebral blood flow (rCBF) of a cortex surface monitoring area, recording rCBF changes before blocking, after blocking and after reperfusion, and detecting that the cortical rCBF suddenly drops to below 15% before blocking by the LDF, thereby indicating that the model of the ischemic stroke model is successfully manufactured. The sham group performed the same procedure, but did not block the middle cerebral artery. During the operation and the recovery period, heat preservation measures are taken to keep the temperature of the anus of the nude mouse between 37 and 37.5 ℃.

2. Ischemic brain region targeted small animal imaging

The near-infrared fluorescence of the heptamethine indocyanine IS used for marking an IS-nSiO preparation. 20 MCAO nude mice are randomly divided into 2 groups (n = 10), the MCAO group IS not treated, the MCAO + IS-nSiO group IS respectively injected with an IS-nSiO preparation from the tail vein of the nude mice, a small animal imager IS placed, isoflurane IS used for maintaining anesthesia, the distribution condition of fluorescence labeling mitochondria in the nude mice brain IS dynamically observed by taking 640nm as an excitation wavelength and 659nm as an emission wavelength, and the time point of strongest fluorescence of the brain IS determined. And then, randomly dividing 20 MCAO nude mice into 2 groups (n = 10), administrating the medicine as above, killing the nude mice at the time point of the strongest fluorescence of IS-nMito, obtaining important organs, and taking near-infrared fluorescence photographs of isolated organs to further determine the brain targeting function of the IS-nMito.

As shown in fig. 5, 5min after IS-nMito administration occurred in the right infarcted brain region, peaking at 30min.

3. Brain tissue fluorescence section mitochondria localization

20 MCAO rats were randomly divided into 2 groups (n = 10), administered separately as above (labeled with MitoTracker), decapitated and brain tissue isolated, and after sectioning the brain tissue, distribution of fluorescently labeled mitochondria of the brain tissue was observed under a fluorescence microscope.

As shown in figure 6, MCAO model was prepared, right middle cerebral artery was blocked for 90min and then opened, after 3d of ischemia reperfusion, the brain slices were cut and fixed, and stained with TTC, dark area representing normal tissue and white area representing ischemic tissue, and the results showed that the infarct volume in IS-nMito group was significantly lower than that in untreated MCAO group, indicating that IS-nMito group could reduce infarct volume.

The above disclosure is only for the specific embodiments of the present invention, but the embodiments of the present invention are not limited thereto, and any variations that can be considered by those skilled in the art should fall within the scope of the present invention.

Claims

1. A preparation method of targeted nanowire mitochondria in an ischemic brain region is characterized by comprising the following steps:

mixing the SB-amylose with carboxyl modified triphenylphosphine for condensation reaction to obtain TPP-SB-amylose;

and (3) extracting nano mitochondria from in vitro blood, mixing the TPP-SB-amylose-Lf-SHp with the nano mitochondria, and performing self-assembly to obtain the ischemic brain region targeted nano mitochondria.

2. The production method according to claim 1,

the mass ratio of the 3-dimethyl (chloropropyl) ammonium propanesulfonate to the amylose is 1: 30-60;

the mass ratio of the TPP-SB-hydroformylation amylose to the homing peptide to the lactoferrin is 10-20: 1;

the mol ratio of the TPP-SB-amylose-Lf-SHp to the nano mitochondria is 1-20: 1.

3. The production method according to claim 2,

the hydrophilic modification is that the stirring is continuously carried out for 6 hours at the temperature of 60 ℃;

the TPP-SB-hydroformylation amylose is connected with the amino groups on the homing peptide and the lactoferrin, namely the lactoferrin and the homing peptide are added into the TPP-SB-hydroformylation amylose solution and incubated for 3-5h at 4 ℃.

4. The preparation method according to claim 3, wherein the specific operation process for extracting the nano mitochondria is as follows: taking isolated blood, carrying out multiple differential centrifugal separation to extract mitochondria, and then sequentially carrying out graded filtration by using 0.45 mu m and 0.22 mu m cell filters to obtain the nano mitochondria with the grain diameter less than 200nm.

5. The preparation method according to claim 1, characterized in that the specific operating steps of the self-assembly are: mixing the nano mitochondria and TPP-SB-amylose-Lf-SHp, magnetically stirring for 30-60min at 4 ℃, and performing centrifugal separation by using a mitochondria buffer solution to obtain the ischemic brain region targeted nano mitochondria;

the mitochondrial buffer solution is prepared from the following raw materials in parts by weight: 0.5 part of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, 2 parts of mannitol, 70 parts of sucrose, 5 parts of 3- (N-morphinone) propanesulfonic acid, 1000 parts of water, and pH7.4.

6. An ischemic brain region-targeted nanowire mitochondria prepared according to the method of any one of claims 1 to 5.

7. Use of the ischemic brain region targeted nanowire mitochondria of claim 6 in the preparation of a medicament for targeting an ischemic brain region.

8. Use of the targeted nanowire mitochondria of the ischemic brain region of claim 6 in the preparation of a medicament for brain repair.

9. A medicament comprising the ischemic brain region targeted nanowire mitochondria of claim 6.