ROS (reactive oxygen species) responsive platelet liposome nanoparticles as well as preparation method and application thereof

Abstract

The invention provides ROS responsive platelet liposome nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The ROS responsive platelet liposome nano-particle comprises a bimolecular layer and deferoxamine wrapped by the bimolecular layer, lycopene is embedded in the middle of the bimolecular layer, and the outer surface of the bimolecular layer is modified with RVG29 polypeptide; the bilayer comprises liposome or is obtained by compounding the liposome and a platelet membrane; the lipidosome is prepared from lecithin, cholesterol, DSPE-PEG2000 (Distearoyl Phosphate Polyethylene Glycol 2000)-MAL (Metallic Acid) and DSPE-Se-Se-PEG2000. The nano-drug is applied as a post-healing drug for treating cerebral arterial thrombosis after thrombolysis, and can be used for treating damage to surrounding tissues after thrombosis of various organs and blood vessels.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to ROS-responsive platelet liposome nanoparticles and a preparation method and application thereof. Background Art

[0002] Stroke is a disease with a high mortality rate and is most likely to cause disability worldwide. Clinically, stroke includes ischemic stroke and ischemia-reperfusion. Common thrombolytic methods for ischemic stroke include tissue plasminogen activator (tPA) thrombolysis and mechanical thrombus removal. In addition, although thrombolytic therapy after ischemia is very important, it can easily cause reperfusion injury. Faced with the oxidative stress caused by reperfusion and the "cascade" outbreak of inflammatory factors, there is currently no more effective clinical treatment.

[0003] Studies have found that patients with ischemic stroke have obvious iron deposition in the brain. The levels of free iron and ferritin in the ischemic side of the brain tissue of the mouse model of cerebral ischemia-reperfusion were significantly increased. Excessive iron will generate a large number of free radicals in the brain tissue, destroying the structure of DNA, RNA and protein of nerve cells, and causing apoptosis of nerve cells. As one of the common iron chelators, deferoxamine (DFO) has the characteristics of rapid chelation of iron in the body and does not compete for hemoglobin iron in blood cells. It has been widely used in the treatment of iron poisoning in clinical practice. However, DFO has a short half-life and is not easy to cross the BBB, so it is difficult to use it to treat iron overload in the central nervous system. Lycopene is a carotenoid with an antioxidant capacity that is twice that of β-carotene and 100 times that of vitamin E. However, it is unstable to light and oxygen and is easily oxidized. Studies have found that lycopene is mainly distributed in the liver and adipose tissue, but not in the brain, which means that lycopene cannot cross the BBB into the brain. Therefore, DFO and lycopene cannot effectively reduce nerve damage caused by ischemia-reperfusion due to the low drug utilization efficiency. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a ROS-responsive platelet liposome nanoparticle, which uses liposome platelets with RVG29 polypeptide with brain microvascular endothelial cell targeting function connected to the surface as a carrier to encapsulate lycopene and deferoxamine, thereby achieving brain delivery of drugs, and at the same time being able to respond to ROS reactions to achieve the purpose of rapid drug release, thereby providing a new means for the treatment of stroke.

[0005] The present invention provides a ROS-responsive platelet liposome nanoparticle, comprising a bilayer and deferoxamine wrapped by the bilayer;

[0006] Lycopene is embedded in the middle of the bilayer, and the outer surface of the bilayer is modified with RVG29 polypeptide;

[0007] The bilayer includes liposomes or is obtained by combining liposomes and platelet membranes;

[0008] The liposome is prepared from lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000.

[0009] Preferably, the RVG29 polypeptide is linked to the maleimide bond of DSPE-PEG2000-MAL in the liposome via cysteine.

[0010] Preferably, the mass ratio of the RVG29 polypeptide to DSPE-PEG2000-MAL is 1:1.

[0011] Preferably, the mass ratio of lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000 is 14:2:1:1.

[0012] Preferably, when the bilayer is obtained by compounding the liposome and the platelet membrane, 1×10 8 ~2×10 8 Platelet membrane.

[0013] Preferably, the concentration of lycopene is 0.7 to 1.5 mg / mL;

[0014] The concentration of the deferoxamine is 2-5 mg / mL.

[0015] The present invention provides a method for preparing the ROS-responsive platelet liposome nanoparticles, comprising the following steps:

[0016] Phosphatidylcholine, cholesterol, DSPE-PEG2000-MAL, DSPE-Se-Se-PEG2000, lycopene and deferoxamine are mixed to obtain liposomes loaded with lycopene and deferoxamine;

[0017] Under the protection of inert gas, the lycopene and deferoxamine loaded liposomes and the cysteine-modified RVG29 polypeptide are subjected to a connection reaction to obtain lycopene and deferoxamine loaded liposomes with the surface modified with RVG29 polypeptide, which are denoted as RSNP-LycoD;

[0018] When the bilayer also includes a platelet membrane, the method further includes mixing the lycopene and deferoxamine loaded liposomes modified with RVG29 polypeptide on the surface with platelets, and co-extruding them to obtain ROS responsive platelet liposome nanoparticles, RSPNP-LycoD nanoparticles.

[0019] The present invention provides the use of the ROS-responsive platelet liposome nanoparticles or the ROS-responsive platelet liposome nanoparticles prepared by the preparation method in the preparation of a drug for preventing and / or treating nerve damage caused by iron overload and redox imbalance. Preferably, the nerve damage caused by iron overload and redox imbalance includes nerve damage caused by reperfusion after thrombolysis or surgical thrombectomy in ischemic stroke.

[0020] Preferably, the dosage form of the drug includes at least one of the following: intravenous injection, capsule, oral solution, nasal spray and pills.

[0021] The invention provides a ROS-responsive platelet liposome nanoparticle, comprising a bilayer and deferoxamine wrapped by the bilayer; lycopene is embedded in the middle of the bilayer, and the outer surface of the bilayer is modified with RVG29 polypeptide; the bilayer comprises a liposome or is obtained by compounding the liposome and a platelet membrane; the liposome is prepared by lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000. The present invention uses the bilayer as the active pharmaceutical ingredients lycopene and deferoxamine, and targets the brain microvascular endothelial cells through the RVG29 polypeptide modified on the outer surface of the bilayer, thereby passing through the blood-brain barrier and delivering the active pharmaceutical ingredients lycopene and deferoxamine to the brain microvascular area. At the same time, the PEG2000-Se-Se-DSPE in the liposome will respond to the ROS of ischemic stroke, and the structure will break under the reaction of ROS, so that the structure of the liposome becomes loose, and it is easier to release drugs in the penumbra area of ​​stroke injury rich in ROS, thereby achieving the purpose of precise targeted drug delivery. At the same time, the present invention also defines that the bimolecule also includes a technical solution formed by the compounding of liposomes and platelet membranes. Since there will be brain microvascular damage in the ischemic area of ​​cerebral stroke, platelets have the natural tendency to damage microvessels and bind to them, and platelets have a natural ability to escape the immune system. Therefore, the tropism of platelets and targeting peptides is utilized to deliver the nanodrug to the penumbra of the stroke. By responding to ROS in the penumbra, the lycopene and DFO in the nanodrug are rapidly released, ultimately solving the problem of accurately targeting the nanodrug to the stroke site and increasing the drug concentration. In the embodiment of the present invention, the MCAO model of the middle cerebral artery occlusion in mice is taken as an example to observe the therapeutic effect of RSPNP-LycoD on cerebral ischemia-reperfusion injury, and it is confirmed that the RSPNP-LycoD nanodrug prepared by the present invention significantly reduces the oxidative stress level and iron level in the ischemia-reperfusion penumbra, and reduces the area of ​​cerebral infarction by more than 2.5 times, providing a new strategy for post-thrombolytic treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The statistical results of particle size of responsive targeted peptide platelet liposomes (RSPNP);

[0023] Figure 2 The results of zeta potential measurement of responsive targeting peptide platelet liposomes (RSPNP);

[0024] Figure 3 The transmission electron microscopy image of responsive platelet-targeted peptide liposome (RSPNP);

[0025] Figure 4 To preserve the test results for the long-term stability of responsive platelet-targeted peptide liposomes (RSPNP);

[0026] Figure 5 The results of in vitro release and reactive oxygen species response curve of co-delivery responsive targeting peptide platelet liposomes (RSPNP-LycoD);

[0027] Figure 6 The peptide connection results of the responsive targeting peptide platelet liposome RSPNP;

[0028] Figure 7 The results of the analysis of the platelet membrane protein component of RSPNP;

[0029] Figure 8 The results of the uptake of RSPNP nanomaterials by neurons and brain microvascular endothelial cell lines (microscope observation);

[0030] Fig. 9 The results of RSPNP-LycoD acting on I / R-induced brain injury in mice;

[0031] Fig.10 The results show that RSPNP targets the ischemic injury area in mice. DETAILED DESCRIPTION

[0032] The invention provides a ROS-responsive platelet liposome nanoparticle, comprising a bilayer and deferoxamine wrapped by the bilayer; lycopene is embedded in the middle of the bilayer, and the outer surface of the bilayer is modified with RVG29 polypeptide; the bilayer comprises a liposome or is obtained by compounding the liposome and a platelet membrane; the liposome is prepared by lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000.

[0033] In the present invention, lycopene and deferoxamine are active pharmaceutical ingredients in the ROS-responsive platelet liposome nanoparticles. As an iron chelator, the deferoxamine can quickly chelate iron in the body and will not compete for hemoglobin iron in blood cells, thereby reducing the iron content in brain tissue, inhibiting the Fenton reaction, and reducing the generation of free radicals. The lycopene has good antioxidant capacity and has a scavenging effect on brain free radicals. The concentration of the lycopene is preferably 0.7-1.5 mg / mL, more preferably 0.9-1.3 mg / mL, and most preferably 1.0 mg / mL. The concentration of the deferoxamine is preferably 2-5 mg / mL, more preferably 3-4 mg / mL, and most preferably 3.5 mg / mL. The deferoxamine is a hydrophilic drug, mixed with the hydrophilic end in the bilayer, and wrapped inside the bilayer. The lycopene is a hydrophobic drug embedded in the middle of the bilayer.

[0034] In the present invention, the bilayer is a carrier of active pharmaceutical ingredients. The RVG29 polypeptide on the bilayer is modified on the liposome. The RVG29 polypeptide is preferably connected to the maleimide bond of DSPE-PEG2000-MAL in the liposome through cysteine. The amino acid sequence of the RVG29 polypeptide is shown in SEQ ID NO:1 (YTIWMPENPRPGTPCDIFTNSRGKRASN), and the RVG29 polypeptide is preferably completed by a method of polypeptide chemical synthesis. The RVG29 polypeptide modified with cysteine ​​preferably adds cysteine ​​during polypeptide chemical synthesis. The thiol group of the cysteine ​​modified by the RVG29 polypeptide is covalently bonded to the maleimide bond of DSPE-PEG2000-MAL, thereby realizing the connection between the RVG29 polypeptide and the liposome. The RVG29 polypeptide is a targeting peptide that can target brain microvascular endothelial cells isolated from rabies virus, which is used to lead nanoparticles to the damaged part of brain microvessels, assist drugs to pass through the blood-brain barrier, thereby improving the delivery of active pharmaceutical ingredients and improving the target concentration of active pharmaceutical ingredients. The mass ratio of the RVG29 polypeptide to DSPE-PEG2000-MAL is preferably 1:1.

[0035] In the present invention, the mass ratio of lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000 in the liposome is preferably 14:2:1:1. The DSPE-PEG2000-MAL is used to connect with the RVG29 polypeptide. The PEG2000-Se-Se-DSPE will respond to ROS in the damaged tissue of ischemic stroke, and the structure will break under the reaction of ROS, making the structure of the liposome loose, making it easier to release drugs in the penumbra of stroke injury rich in ROS.

[0036] In the present invention, when the bilayer is obtained by compounding the liposome and the platelet membrane, preferably, 1×10 8 ~2×10 8 platelet membranes, more preferably 1.5×10 8 The platelet membrane has the natural property of tending to damage microvessels. Since the ischemic area of ​​cerebral stroke may have cerebral microvascular damage, the platelet membrane combined with the polypeptide delivers the active ingredients of the drug to the penumbra of cerebral stroke. By responding to ROS in the penumbra, lycopene and DFO are quickly released, which ultimately solves the problem of accurately targeting nanomedicine to the ischemic stroke site and increasing the drug concentration.

[0037] The present invention provides a method for preparing the ROS-responsive platelet liposome nanoparticles, comprising the following steps:

[0038] Phosphatidylcholine, cholesterol, DSPE-PEG2000-MAL, DSPE-Se-Se-PEG2000, lycopene and deferoxamine are mixed to obtain liposomes loaded with lycopene and deferoxamine;

[0039] Under the protection of inert gas, the lycopene and deferoxamine loaded liposomes and the cysteine-modified RVG29 polypeptide are subjected to a connection reaction to obtain lycopene and deferoxamine loaded liposomes with the surface modified with RVG29 polypeptide, which are denoted as RSNP-LycoD;

[0040] When the bilayer also includes a platelet membrane, the method further includes mixing the lycopene and deferoxamine loaded liposomes modified with RVG29 polypeptide on the surface with platelets, and co-extruding them to obtain ROS responsive platelet liposome nanoparticles, RSPNP-LycoD nanoparticles.

[0041] The present invention mixes lecithin, cholesterol, DSPE-PEG2000-MAL, DSPE-Se-Se-PEG2000, lycopene and deferoxamine to obtain liposomes loaded with lycopene and deferoxamine, which are recorded as SNP-LycoD.

[0042] In the present invention, after the mixing, preferably rotary evaporation is included. The temperature of the rotary evaporation is preferably 30-40° C., more preferably 35° C. The time of the rotary evaporation is preferably 12-17 min, more preferably 15 min.

[0043] After obtaining the liposomes loaded with lycopene and deferoxamine, the present invention, under the protection of inert gas, carries out a connection reaction between the liposomes loaded with lycopene and deferoxamine and the cysteine-modified RVG29 polypeptide to obtain the liposomes loaded with lycopene and deferoxamine whose surface is modified with the RVG29 polypeptide, which is recorded as RSNP-LycoD.

[0044] In the present invention, the temperature of the ligation reaction is preferably 20-30° C., more preferably 25° C. The time of the ligation reaction is preferably 15-30 min, more preferably 20 min. The inert gas is preferably nitrogen.

[0045] When the bilayer of the present invention also includes a platelet membrane, the present invention further includes mixing the liposomes loaded with lycopene and deferoxamine and modified with RVG29 polypeptide on the surface with platelets, and co-extruding them to obtain ROS-responsive platelet liposome nanoparticles, RSPNP-LycoD nanoparticles.

[0046] In the present invention, the co-extrusion is preferably completed with a liposome extruder. In vitro experiments show that the particle size of the ROS-responsive platelet liposome nanoparticles is 134-135nm, which is a nanoparticle that can penetrate the blood-brain barrier (BBB). The average potential of the ROS-responsive platelet liposome nanoparticles is -40--41mV. Transmission electron microscopy observed that the morphology of the ROS-responsive platelet liposome nanoparticles was a round sphere. Long-term stability experiments show that the ROS-responsive platelet liposome nanoparticles have good stability and can be stored in PBS at 4°C for at least 96 hours. The nanomedicine can be relatively stable in a culture medium containing 10% FBS at 37°C for 72 hours. At the same time, the in vitro release and reactive oxygen response curve results of RSPNP-LycoD show that the release rate of DFO in PBS treated with reactive oxygen is 79.04%; the release rate of tomato red in PBS treated with reactive oxygen is 68.89%. It shows that the nanomedicine responds to reactive oxygen and will quickly release the drug under the influence of a large amount of reactive oxygen in the stroke area. Confocal microscopy showed that the peptide could bind to liposomes and mediate the entry of liposomes into cells. Analysis of platelet membrane protein components showed that RSPNP-LycoD had protein bands similar to those of platelets, and had platelet-specific CD47, CD41 and CD36 proteins, and had functions similar to those of platelet membranes. The uptake experiment of RSPNP nanomaterials by neurons and brain microvascular endothelial cells showed that both neurons and brain microvascular endothelial cells were more inclined to uptake RSPNP-LycoD nanomaterials, and the uptake was inhibited by peptide pretreatment.

[0047] In the present invention, a method for verifying the ROS response of ROS-responsive platelet liposome nanoparticles is provided. After a semipermeable membrane containing a ROS-responsive platelet liposome nanoparticle sample is tightly sealed, the semipermeable membrane is placed in normal deionized water and 200 μM hydrogen peroxide for incubation, and the drug content outside the semipermeable membrane is detected. When drugs are released outside the semipermeable membrane in hydrogen peroxide, but no drugs are released outside the semipermeable membrane in deionized water, it indicates that the ROS-responsive platelet liposome nanoparticles undergo structural changes in response to ROS.

[0048] In the present invention, RSPNP-LycoD nanomedicine was used to treat an ischemic stroke mouse model, and the results showed that RSPNP-LycoD significantly reduced the infarct area compared with other treatment groups, indicating that RSPNP-LycoD has a significant neuroprotective effect on cerebral ischemia-reperfusion damage.

[0049] The present invention provides the use of the ROS-responsive platelet liposome nanoparticles or the ROS-responsive platelet liposome nanoparticles prepared by the preparation method in preparing a drug for preventing and / or treating nerve damage caused by iron overload and redox imbalance.

[0050] In the present invention, the nerve damage caused by iron overload and redox imbalance preferably includes nerve damage caused by reperfusion after thrombolysis or surgical thrombectomy of ischemic stroke. The ROS-responsive platelet liposome nanoparticles can be used as a post-healing drug for the treatment of thrombolysis of ischemic stroke, and can be used to treat damage to surrounding tissues caused by thrombosis of various organs and blood vessels.

[0051] In the present invention, the dosage form of the drug preferably includes at least one of the following: intravenous injection, capsule, oral solution, nasal spray and dripping pill. The present invention has no particular limitation on the preparation method of the drug, and the preparation method of the drug known in the art can be used.

[0052] The following is a detailed description of a ROS-responsive platelet liposome nanoparticle provided by the present invention, and its preparation method and application, in conjunction with the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] Preparation of co-delivery responsive targeting peptide platelet liposomes (RSPNP-LycoD)

[0055] Step 1: Preparation of platelet membrane

[0056] 1. Reagent preparation:

[0057] 1.1 HEPE buffer:

[0058] NaCl: KCl: HEPES: EGTA (a calcium ion chelator) were dissolved in water at a mass ratio of 1: 0.02: 0.1: 0.2, and the pH was adjusted to 7.4 to prepare a HEPE buffer;

[0059] 1.2 Wash buffer:

[0060] Dissolve NaCl, EDTA, dextrose, and sodium citrate in water at a mass ratio of 1:0.02:1.0:0.3, and adjust the pH to 7.6 to prepare a washing buffer;

[0061] 1.3 Tyrode's buffer:

[0062] NaCl, NaHCO3, KCl, Na2HPO4, MgCl2, and HEPES were dissolved in water at a mass ratio of 1:0.1:0.02:0.005:0.01:0.3, and then PEG, PMSF, and water were added to PEG and PMSF at a volume ratio of 1:1:10000 and the pH was adjusted to 7.5 to prepare a benchtop buffer.

[0063] 2. Preparation process:

[0064] (1) Adult mice were selected, blood was collected by cardiac sampling, and anticoagulant was added to collect the blood into a tube;

[0065] (2) Blood was processed and platelets were collected within 2 h after collection, and whole blood was further anticoagulated with HEPE buffer at a ratio of 10:1;

[0066] (3) Centrifuge at 100 g for 15 min and collect the supernatant to obtain platelet-rich plasma;

[0067] (4) Centrifuge at 1000 g for 16 min and collect the precipitate;

[0068] (5) Add red blood cell lysis buffer (5:1), lyse at low temperature for 5 min, and repeat step (4);

[0069] (6) Add wash buffer to resuspend the precipitate and repeat step (4);

[0070] (7) Resuspend the precipitate with Tyrode's buffer or water and count the collected platelets using a platelet counter;

[0071] (8) Repeated freezing and thawing for 3 times, -80°C to room temperature cycle, and ultrasonic disruption to obtain platelet membrane (PNV);

[0072] Step 2: Preparation of RSPNP-LycoD

[0073] (1) Add lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. Then add 200 μL of 10 mg / mL lycopene (dissolved in tetrahydrofuran). After mixing evenly, add 2 mL of 10 mg / mL DFO (dissolved in phosphate buffer) to the mixture. In a 35°C water bath, at a speed of 80 r / min, rotary evaporate for 10 min until the liquid is concentrated to 2 mL, to obtain responsive co-delivery lycopene and deferoxamine liposomes (SNP-LycoD);

[0074] (2) The liposomes were stirred and reacted with 1 mg of RVG29-Cys peptide for 20 min under nitrogen flow;

[0075] (3) Add about 1.0×10 8 The PNV membrane made of platelets was co-extruded through a liposome extruder, and then extruded through 400 nm and 200 nm filter membranes in sequence, and RSPNP-LycoD was obtained after repeated extrusion;

[0076] (4) The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) at 4°C, 3000 g, and ultrafiltered for 60 min. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-LycoD was finally obtained.

[0077] The encapsulation efficiency of lycopene and deferoxamine in the obtained RSPNP-LycoD nanomedicine was determined by the following method:

[0078] A. Lycopene detection method

[0079] Take 100 μL of the liposome solution to be measured and add it to 1 mL of tetrahydrofuran. Ultrasonicate for 10 minutes to destroy the liposome structure. Centrifuge at 12000g for 10 minutes at room temperature. Aspirate the supernatant and make up to 2 mL with tetrahydrofuran. Calculate the OD value under 452nm light conditions under ultraviolet spectrophotometer detection. Substitute the OD values ​​of different concentrations of standard products under 452nm light conditions to establish a standard curve (see formula I). ​​After calculation, the lycopene content is obtained, and the encapsulation rate of lycopene is calculated according to formula II.

[0080] The standard curve is: y=0.0791x-0.0016 Formula I.

[0081] Among them, R 2 =0.9996, y is the concentration of lycopene, and x is the detected OD value.

[0082]

[0083] B. Deferoxamine detection method

[0084] Take 100 μL of the lower layer solution of the ultrafiltration tube in step 2 (4), add the prepared acetic acid-sodium acetate buffer (HAc-NaAc, pH=6.5) until the volume is 4.5 mL. Then add 0.5 mL of 1 mM ferrous sulfate solution and react in the dark for 10 minutes. Under ultraviolet spectrophotometer detection, calculate the OD value under 425 nm light conditions. Calculate the OD values ​​of different concentrations of standards under 425 nm light conditions to establish a standard curve (see formula III). Reversely calculate the deferoxamine content in the liposomes, and calculate the encapsulation rate of deferoxamine according to formula IV.

[0085] The standard curve is: y=2.4871x-0.0425 Formula III.

[0086] R 2 =0.9884, y is the concentration of deferoxamine, and x is the detected OD value.

[0087]

[0088] In the RSPNP-LycoD nanomedicine prepared in this example, the encapsulation efficiency of lycopene is 60.27%, and the encapsulation efficiency of deferoxamine is 30.2%.

[0089] Step 3: Detection of the release rate and ROS response level of nanomedicine.

[0090] (1) The nanomedicine obtained in step 1 was divided into four portions, and each portion was placed in an activated dialysis bag (Solarbio, Cat: YA1078), tied with cotton thread, and lightly pressed to detect whether the dialysis bag leaked.

[0091] (2) Place the dialysis bag in a 50 mL centrifuge tube containing 30 mL of phosphate buffer and a prepared 200 μM hydrogen peroxide solution, as well as a phosphate buffer with 0.02% Tween 20 and a 200 μM hydrogen peroxide solution with 2% Tween 20. Cover the lid to ensure it is completely sealed.

[0092] (3) Fix the centrifuge tube on a constant temperature shaker, set the shaker to 37°C and the shaking frequency to 120 rpm / min.

[0093] (4) After incubation for 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h, 1 mL of the solution outside the dialysis bag was taken out and the corresponding solution was added. After the samples were collected, the lycopene and deferoxamine content in the samples were detected according to step 2.

[0094] (5) Calculate the release efficiency of lycopene and deferoxamine according to formula V:

[0095]

[0096] Among them, E r : Cumulative drug release; V e : replacement volume of the solution outside the dialysis bag; V0: volume of the solution outside the dialysis bag (30 mL); C i : The concentration of the drug in the external solution at the time of the i-th replacement sampling; m drug : the total mass of nano-drug loading; n: the number of sampling; C n : After sampling n times, the drug concentration of the external solution finally detected by the method provided in step 2.

[0097] Among them, when calculating deferoxamine, the two groups without Tween 20 were calculated. Due to its own hydrophobicity, lycopene was calculated in the last two groups with Tween 20. The final calculation results are: the release efficiency of deferoxamine in phosphate buffer at 4h, 6h, 8h, 12h, 24h, 48h and 72h was 0.283822, 0.57216, 0.61225, 0.688227, 0.632381, 0.700618 and 0.820648 respectively; the release efficiency in hydrogen peroxide solution was 0.58344, 0.708441, 0.773629, 0.699486, 0.699465, 0.945032 and 0.895518. The release efficiency of lycopene in phosphate buffer was 0.373602, 0.342744, 0.501506, 0.57041, 0.647087, 0.681506 and 0.632984; and the release efficiency in hydrogen peroxide solution was 0.611812, 0.736681, 0.69379, 0.672239, 0.659067, 0.734574 and 0.576296.

[0098] Example 2

[0099] Preparation of RSPNP-LycoD nanodrugs (encapsulation efficiency: lycopene 56.91% deferoxamine 28.12%)

[0100] Step 1: Preparation of platelet membrane

[0101] 1. Reagent preparation:

[0102] 1.1 HEPE buffer:

[0103] NaCl, KCl, HEPES, and EGTA were dissolved in water at a mass ratio of 1:0.02:0.1:0.2, and the pH was adjusted to 7.4 to prepare a HEPES buffer;

[0104] 1.2 Wash buffer:

[0105] Dissolve NaCl, EDTA, dextrose and sodium citrate in water in a mass ratio of 1:0.03:1.0:0.4 and adjust the pH to 7.6 to prepare a washing buffer;

[0106] 1.3 Tyrode's buffer:

[0107] NaCl, NaHCO3, KCl, Na2HPO4, MgCl2, and HEPES were dissolved in water at a mass ratio of 1:0.2:0.04:0.007:0.02:0.4, and then PEG and PMSF were added to water at a volume ratio of 1:1:10000 and the pH was adjusted to 7.5 to prepare a benchtop buffer;

[0108] 2. Preparation process:

[0109] (1) Adult mice were selected, blood was collected by cardiac sampling, and anticoagulant was added to collect the blood into a tube;

[0110] (2) Blood was processed and platelets were collected within 0.5 h after collection, and whole blood was further anticoagulated with HEPE buffer at a ratio of 10:1;

[0111] (3) Centrifuge at 100 g for 15 min and collect the supernatant to obtain platelet-rich plasma;

[0112] (4) Centrifuge at 1000 g for 18 min and collect the precipitate;

[0113] (5) Add red blood cell lysis buffer (5:1), lyse at low temperature for 5 min, and repeat step (4);

[0114] (6) Add wash buffer to resuspend the precipitate and repeat step (4);

[0115] (7) Resuspend the precipitate with Tyrode's buffer or water and count the collected platelets using a platelet counter;

[0116] (8) Repeated freezing and thawing for 3 times, -80°C to room temperature cycle, and ultrasonic disruption to obtain platelet membrane (PNV);

[0117] Step 2: Preparation of RSPNP-LycoD

[0118] (1) Add lecithin: cholesterol: DSPE-PEG2000-MAL: DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. Then add 200 μL of 10 mg / mL lycopene (dissolved in tetrahydrofuran). After mixing evenly, add 2 mL of 10 mg / mL DFO (dissolved in phosphate buffer) to the mixture. In a 35°C water bath, the speed is 70 r / min, and the rotary evaporation is carried out for 12 minutes until the liquid is concentrated to about 1-2 mL. Responsive co-delivery of lycopene and deferoxamine liposomes (SNP-LycoD) are obtained;

[0119] (2) The liposomes were stirred and reacted with 1 mg of RVG29-Cys peptide for 20 min under nitrogen flow;

[0120] (3) Add about 1.5×10 8 The PNV membrane made of platelets was co-extruded through a liposome extruder, and then extruded through 400 nm and 200 nm filter membranes in sequence, and RSPNP-LycoD was obtained after repeated extrusion;

[0121] (4) The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) at 4°C, 3000 g, and ultrafiltered for 60 min. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-LycoD was finally obtained.

[0122] The encapsulation efficiency of the RSPNP-LycoD was determined according to the method of Example 1.

[0123] The lycopene encapsulation rate in the RSPNP-LycoD nano drug is 56.91%, and the deferoxamine encapsulation rate is 28.12%.

[0124] Step 3: Detection of the release rate and ROS response level of nanomedicine.

[0125] (1) The nanomedicine obtained in step 1 was divided into four portions, and each portion was loaded into an activated dialysis bag (Solarbio, Cat: YA1078), tied with cotton thread, and lightly pressed to detect whether the dialysis bag leaked.

[0126] (2) Place the dialysis bag in a 50 mL centrifuge tube containing 30 mL of phosphate buffer and a prepared 200 μM hydrogen peroxide solution, as well as a phosphate buffer with 0.02% Tween 20 and a 200 μM hydrogen peroxide solution with 2% Tween 20. Cover the lid to ensure it is completely sealed.

[0127] (3) Fix the centrifuge tube on a constant temperature shaker, set the shaker to 37°C and the shaking frequency to 120 rpm / min.

[0128] (4) After incubation for 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h, 1 mL of the solution outside the dialysis bag was taken out and the corresponding solution was added. After the samples were collected, the lycopene and deferoxamine content in the samples were detected according to step 2.

[0129] (5) The release efficiency of lycopene and deferoxamine was calculated according to formula III.

[0130] Among them, when calculating deferoxamine, the two groups without Tween 20 were calculated. Due to its own hydrophobicity, lycopene was calculated in the last two groups with Tween 20. The final calculation results are: the release efficiency of deferoxamine in phosphate buffer at 4h, 6h, 8h, 12h, 24h, 48h and 72h was 0.165225, 0.573464, 0.701324, 0.678091, 0.693105, 0.832346 and 0.830337 respectively; the release efficiency in hydrogen peroxide solution was 0.689184, 0.745319, 0.776029, 0.826858, 0.752556, 0.867277 and 0.83918. The release efficiency of lycopene in phosphate buffer was 0.416522, 0.419981, 0.505118, 0.573065, 0.681329, 0.521674 and 0.670942; and the release efficiency in hydrogen peroxide solution was 0.6, 0.61583, 0.531442, 0.681112, 0.742337, 0.724309 and 0.802196.

[0131] Example 3

[0132] Preparation of RSPNP-LycoD nanomedicine

[0133] Step 1: Preparation of platelet membrane

[0134] 1. Reagent preparation:

[0135] 1.1 HEPE buffer:

[0136] NaCl, KCl, HEPES, and EGTA were dissolved in water at a mass ratio of 1:0.02:0.1:0.2, and the pH was adjusted to 7.4 to prepare a HEPES buffer;

[0137] 1.2 Wash buffer:

[0138] Dissolve NaCl, EDTA, dextrose, and sodium citrate in water at a mass ratio of 1:0.03:1.0:0.3, and adjust the pH to 7.2 to prepare a washing buffer;

[0139] 1.3 Tyrode's buffer:

[0140] NaCl, NaHCO3, KCl, Na2HPO4, MgCl2, and HEPES were dissolved in water at a mass ratio of 1:0.1:0.02:0.005:0.01:0.3, and then PEG and PMSF were added to water at a volume ratio of 1:1:10000 and the pH was adjusted to 7.4 to prepare a benchtop buffer;

[0141] 2. Preparation process:

[0142] (1) Adult mice were selected, blood was collected by cardiac sampling, and anticoagulant was added to collect the blood into a tube;

[0143] (2) Blood was processed and platelets were collected within 1 h after collection, and whole blood was further anticoagulated with HEPE buffer at a ratio of 10:1;

[0144] (3) Centrifuge at 100 g for 17 min and collect the supernatant to obtain platelet-rich plasma;

[0145] (4) Centrifuge at 1000 g for 15 min and collect the precipitate;

[0146] (5) Add red blood cell lysis buffer (5:1), lyse at low temperature for 5 min, and repeat step (4);

[0147] (6) Add wash buffer to resuspend the precipitate and repeat step (4);

[0148] (7) Resuspend the precipitate with Tyrode's buffer or water and count the collected platelets using a platelet counter;

[0149] (8) Repeated freezing and thawing for 3 times, -80°C to room temperature cycle, and ultrasonic disruption to obtain platelet membrane (PNV);

[0150] Step 2: Preparation of RSPNP-LycoD

[0151] (1) Add lecithin: cholesterol: DSPE-PEG2000-MAL: DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. Then add 200 μL of 10 mg / mL lycopene (dissolved in tetrahydrofuran). After mixing evenly, add 2 mL of 10 mg / mL DFO (dissolved in phosphate buffer) to the mixture. In a 35°C water bath, at a speed of 80 r / min, rotary evaporate for 20 minutes until the liquid is concentrated to about 1-2 mL. Responsive co-delivery of lycopene and deferoxamine liposomes (SNP-LycoD) are obtained;

[0152] (2) The liposomes were stirred and reacted with 1 mg of RVG29-Cys peptide for 20 min under nitrogen flow;

[0153] (3) Add about 2×10 8 The PNV membrane made of platelets was co-extruded through a liposome extruder, and then extruded through 400 nm and 200 nm filter membranes in sequence, and RSPNP-LycoD was obtained after repeated extrusion;

[0154] (4) The collected liposomes were placed in an ultrafiltration centrifuge tube (10kDa) at 4°C, 3000g, and ultrafiltration was performed for 60 minutes. After completion, 1 mL of PBS was added to the upper ultrafiltration tube, and after being blown and aspirated evenly, ultrafiltration was continued for 60 minutes. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-LycoD was finally obtained. The lycopene encapsulation efficiency of the obtained RSPNP-LycoD nanomedicine was 74.51%, and the deferoxamine encapsulation efficiency was 26.51%.

[0155] Step 3: Detection of the release rate and ROS response level of nanomedicine.

[0156] (1) The nanomedicine obtained in step 1 was divided into four portions, and each portion was loaded into an activated dialysis bag (Solarbio, Cat: YA1078), tied with cotton thread, and lightly pressed to detect whether the dialysis bag leaked.

[0157] (2) Place the dialysis bag in a 50 mL centrifuge tube containing 30 mL of phosphate buffer and a prepared 200 μM hydrogen peroxide solution, as well as a phosphate buffer with 0.02% Tween 20 and a 200 μM hydrogen peroxide solution with 2% Tween 20. Cover the lid to ensure it is completely sealed.

[0158] (3) Fix the centrifuge tube on a constant temperature shaker, set the shaker to 37°C and the shaking frequency to 120 rpm / min.

[0159] (4) After incubation for 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h, 1 mL of the solution outside the dialysis bag was taken out and the corresponding solution was added. After the samples were collected, the lycopene and deferoxamine content in the samples were detected according to step 2.

[0160] (5) The release efficiency of lycopene and deferoxamine was calculated according to formula III.

[0161] Among them, when calculating deferoxamine, the two groups without Tween 20 were calculated. Due to its own hydrophobicity, lycopene was calculated in the latter two groups with Tween 20. The final calculation results are: the release efficiency of deferoxamine in phosphate buffer at 4h, 6h, 8h, 12h, 24h, 48h and 72h was 0.47085, 0.613209, 0.640163, 0.681425, 0.758575, 0.746123 and 0.709766 respectively; the release efficiency in hydrogen peroxide solution was 0.731214, 0.713367, 0.771517, 0.844915, 0.898843, 0.911489 and 0.818113. The release efficiency of lycopene in phosphate buffer was 0.467825, 0.363983, 0.428148, 0.374629, 0.476535, 0.545781 and 0.436327; and the release efficiency in hydrogen peroxide solution was 0.512414, 0.637131, 0.624386, 0.713432, 0.711623, 0.812354 and 0.834576.

[0162] Example 4

[0163] The method for preparing ROS-responsive platelet liposomes modified with targeting peptides and loaded with lycopene and deferoxamine comprises the following steps:

[0164] Step 1: Preparation of platelet membrane

[0165] 1. Reagent preparation:

[0166] 1.1 HEPE buffer:

[0167] Dissolve NaCl:KCl:HEPES:EGTA in water at a mass ratio of 1:0.02:0.1:0.2 and adjust the pH to 7.4 to prepare HEPE buffer;

[0168] 1.2 Wash buffer:

[0169] Dissolve NaCl, EDTA, dextrose, and sodium citrate in water at a mass ratio of 1:0.02:1.0:0.3, and adjust the pH to 7.6 to prepare a washing buffer;

[0170] 1.3 Tyrode's buffer:

[0171] NaCl, NaHCO3, KCl, Na2HPO4, MgCl2, and HEPES were dissolved in water at a mass ratio of 1:0.1:0.02:0.005:0.01:0.3, and then PEG, PMSF, and water were added to PEG and PMSF at a volume ratio of 1:1:10000 and the pH was adjusted to 7.5 to prepare a benchtop buffer.

[0172] 2. Platelet membrane preparation process:

[0173] (1) Adult mice were selected, blood was collected by cardiac sampling, and anticoagulant was added to collect the blood into a tube;

[0174] (2) Blood was processed and platelets were collected within 4 h after collection, and whole blood was further anticoagulated with HEPE buffer at a ratio of 10:1;

[0175] (3) Centrifuge at 100 g for 15-20 min and collect the supernatant to obtain platelet-rich plasma;

[0176] (4) Centrifuge at 1000 g for 15-20 min and collect the precipitate;

[0177] (5) Add red blood cell lysis buffer (5:1), lyse at low temperature for 5 min, and repeat step (4);

[0178] (6) Add wash buffer to resuspend the precipitate and repeat step (4);

[0179] (7) Resuspend the precipitate with Tyrode's buffer or water and count the collected platelets using a platelet counter;

[0180] (8) Repeated freezing and thawing for 3 times, -80°C to room temperature cycle, and ultrasonic disruption to obtain platelet membrane (PNV);

[0181] Step 2: Preparation of PNP, RPNP and RSPNP

[0182] (1) Preparation of platelet-derived liposomes (PNP)

[0183] a. Add lecithin: cholesterol: DSPE-PEG2000-MAL in a mass ratio of 7:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. After mixing evenly, add 2 mL of phosphate buffer to the mixture. Place in a 30-40℃ water bath, rotate at 70-80 r / min, and evaporating for 10-15 minutes until the liquid is concentrated to about 1-2 mL.

[0184] b. Add about 1.0×10 8 The PNV membrane made of platelets was coextruded through a liposome extruder, and then extruded through 400 nm and 200 nm filters, repeatedly;

[0185] c. The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) and ultrafiltered for 60 min at 4°C, 3000 g. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and PNP-LycoD was finally obtained.

[0186] (2) Preparation of platelet-targeted peptide liposomes (RPNP)

[0187] a. Add lecithin: cholesterol: DSPE-PEG2000-MAL in a mass ratio of 7:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. After mixing evenly, add 2 mL of phosphate buffer to the mixture. Place in a 35°C water bath at a speed of 80 r / min and perform rotary evaporation for 10 minutes until the liquid is concentrated to 1-2 mL.

[0188] b. The liposomes were stirred and reacted with 1 mg of RVG29-Cys peptide under nitrogen.

[0189] c. Add about 1.0×10 8 The PNV membrane made of platelets was coextruded through a liposome extruder, and then extruded through 400 nm and 200 nm filters, repeatedly;

[0190] d. The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) and ultrafiltered for 60 min at 4°C, 3000 g. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RPNP-LycoD was finally obtained.

[0191] (3) Preparation of responsive platelet-targeted peptide liposomes (RSPNP)

[0192] a. Add lecithin: cholesterol: DSPE-PEG2000-MAL: DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. After mixing evenly, add 2 mL of phosphate buffer to the mixture. Place in a 30-40°C water bath, rotate at 80 r / min, and evaporating for 10 minutes until the liquid is concentrated to 1-2 mL. Responsive liposomes for co-delivery of lycopene and deferoxamine are obtained;

[0193] b. The liposomes were reacted with 1 mg of RVG29 polypeptide under stirring while nitrogen was introduced;

[0194] c. Add about 1.0×10 8 The PNV membrane made of platelets was coextruded through a liposome extruder, and then extruded through 400 nm and 200 nm filters, repeatedly;

[0195] d. The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) and ultrafiltered for 60 min at 4°C, 3000 g. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-LycoD was finally obtained.

[0196] Step 3. Preparation of Free-LycoD and RSPNP-Lyco

[0197] (1) Preparation of Free-LycoD

[0198] 1.2 mg of lycopene was dissolved in 200 μL of corn oil, mixed with 5 mg of deferoxamine dissolved in 2 mL of corn oil, and oscillated and ultrasonicated to form a turbid solution to obtain the free drug Free-LycoD

[0199] (2) Preparation of responsive platelet-targeted peptide liposomes (RSPNP-Lyco) for delivering lycopene

[0200] a. Add lecithin: cholesterol: DSPE-PEG2000-MAL: DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. Then add 200 μL of 10 mg / mL lycopene (dissolved in tetrahydrofuran). After mixing evenly, add 2 mL of phosphate buffer to the mixture. In a 35°C water bath, at a speed of 80 r / min, rotary evaporate for 10 minutes until the liquid is concentrated to 1-2 mL. Responsive co-delivery of lycopene and deferoxamine liposomes (SNP-Lyco) are obtained;

[0201] b. The liposomes were reacted with 1 mg of RVG29-Cys polypeptide under stirring under nitrogen to obtain liposome RSNP-Lyco;

[0202] c. Add about 1.0×10 8 The PNV membrane made of platelets was coextruded through a liposome extruder, and then extruded through 400 nm and 200 nm filter membranes in sequence, and RSPNP-Lyco was obtained after repeated extrusion;

[0203] d. The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) and ultrafiltered for 60 min at 4°C, 3000 g. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-Lyco was finally obtained.

[0204] (3) Preparation of co-delivery responsive targeting peptide platelet liposomes (RSPNP-LycoD)

[0205] a. Add lecithin: cholesterol: DSPE-PEG2000-MAL: DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol. Add 200 μL of 10 mg / mL lycopene (dissolved in tetrahydrofuran). After mixing evenly, add 2 mL of 10 mg / mL DFO (dissolved in phosphate buffer) to the mixture. In a 35°C water bath, at a speed of 80 r / min, rotary evaporate for 10 minutes until the liquid is concentrated to 1-2 mL. Responsive co-delivery of lycopene and deferoxamine liposomes (SNP-LycoD) are obtained;

[0206] b. The liposomes were reacted with 1 mg RVG29-Cys polypeptide under stirring while nitrogen was introduced;

[0207] c. Add about 1.0×10 8 The PNV membrane made of platelets was coextruded through a liposome extruder, and then extruded through 400 nm and 200 nm filters, repeatedly;

[0208] d. The collected liposomes were placed in an ultrafiltration centrifuge tube (10 kDa) and ultrafiltered for 60 min at 4°C, 3000 g. After the ultrafiltration, 1 mL of PBS was added to the upper ultrafiltration tube, and after being pipetted evenly, the ultrafiltration was continued for 60 min. Finally, 1 mL of PBS was added to resuspend the upper ultrafiltration tube, and RSPNP-LycoD was finally obtained.

[0209] Example 5

[0210] Example 4 Morphological characterization experiment of prepared responsive peptide platelet-targeted liposome nanomedicine

[0211] 1. Dynamic light scattering technology was used to determine the particle size of responsive targeting peptide platelet liposomes and controls (platelet liposomes and targeting peptide platelet liposomes).

[0212] The results are as follows Figure 1 As shown. The average hydrated particle sizes of platelet-derived liposomes (PNP), platelet-derived peptide-targeted liposomes (RPNP) and platelet-derived responsive peptide-targeted liposomes (RSPNP) are 127.73±1.10nm, 132.00±4.83nm and 134.73±4.19nm. The hydrated particle size of the nanomaterials can penetrate the blood-brain barrier (BBB).

[0213] 2. Zeta potential detection

[0214] Zeta potential is an important indicator for characterizing the stability of dispersion systems. Dynamic light scattering (DLS) was used to measure the Zeta potential of responsive targeting peptide platelet liposome nanomedicine and its control.

[0215] like Figure 2 As shown, the average potentials of the three nanomaterials are -60.10±0.78mV, -41.33±0.82mV and -40.37±0.54mV respectively. The electrical properties of the three nanomaterials are stable and dispersed nanomaterials.

[0216] 3. Observe the responsive targeting peptide platelet liposomes using transmission electron microscopy.

[0217] Figure 3 It shows that the responsive targeting peptide platelet liposome (RSPNP) nanocarrier prepared by the present invention has a regular round sphere shape.

[0218] 4. Stability test

[0219] The responsive targeted peptide platelet liposome nanomedicine and its control were stored in PBS buffer at 4°C and culture medium containing 10% FBS at 37°C, respectively. The particle size changes of the nanomedicine were detected by dynamic light scattering after being stored for 24h, 36h, 48h and 72h.

[0220] like Figure 4The particle sizes of the nanomaterials were 123.33nm, 123.00nm and 144.13nm after 96h storage at 4℃. The particle sizes after 72h storage at 37℃ containing 10% FBS were 137.10nm, 137.93nm and 149.00nm, respectively, with no significant changes. This shows that the nanomedicine can be stored in PBS at 4℃ for at least 96h, and the nanomedicine can be relatively stable in the culture medium containing 10% FBS at 37℃ for 72h.

[0221] 5. Drug active ingredient release experiment and reactive oxygen species response curve

[0222] The RSPNP-LycoD prepared in Examples 1 to 3 was divided into two equal portions and placed in two dialysis bags (Solarbio, YA1078), which were fastened and placed in centrifuge tubes containing PBS and PBS containing 200 μM H2O2, respectively. The incubation was carried out at 37°C and 120 rpm with shaking. Samples were taken at different time points, and the drug release rate was calculated according to the average value. The results are shown in Figure 5 After 12 hours, the release rate of DFO in normal PBS was 68.0%, and in PBS treated with reactive oxygen species, the release rate was 79.04%; the release rate of tomato red in normal PBS was 51.0%, and in PBS treated with reactive oxygen species, the release rate was 68.89%. This indicates that nanomedicines respond to reactive oxygen species and will quickly release drugs under the influence of a large amount of reactive oxygen species in the stroke area.

[0223] 6. Peptide connection of responsive platelet-targeted peptide liposomes (RSPNP)

[0224] Liposome synthesis: Put lecithin, cholesterol, DSPE-PEG2000-MAL, and DSPE-Se-Se-PEG2000 in a mass ratio of 14:2:1:1 into an eggplant-shaped bottle containing 5 mL of anhydrous ethanol dissolved with 1 mg DiO (Yeasen, 40725ES), and mix with 2 mL of phosphate buffer. In a 30-40°C water bath, the speed is 70-80 r / min, and the rotary evaporation is 10-15 minutes until the liquid is concentrated to 1-2 mL. Co-extrude through a liposome extruder, extrude through 400nm and 200nm filter membranes in turn, and extrude repeatedly to obtain liposomes;

[0225] Platelet vesicles: DiD (Beyotime, C1039-10mg), diluted with PBS to 100μM, added to the platelet vesicles extracted in step 1, incubated at 37°C for 20min. Ultrafiltration and concentration were performed through an ultrafiltration tube, and re-collected after dilution with 500μL PBS;

[0226] Fluorescence labeling of peptide RVG29: prepare 1 mg of peptide, 1 mg of sulfo-Cy3-NHS (XINQBIO, 1686147-68-1), and 1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Macklin, 25952-53-8). Activate in EDC / sulfo-Cy3-NHS (pH 6.0) for 20 min, add triethylamine to adjust the pH to above 7.0, and react with the peptide at room temperature for 2 h (nitrogen is introduced for reaction).

[0227] Synthesis of combined responsive targeting peptide platelet liposomes: After the above liposomes were synthesized, 1 mg of the above labeled peptide was added under nitrogen sealing conditions for stirring and reaction at room temperature for 20 minutes. Then, the above labeled platelet vesicles were added and co-extruded under a 400 nm membrane using a liposome extruder.

[0228] The synthesized materials were added to the cell culture medium for incubation. Liposomes, platelet vesicles and peptides were added to one dish. The combined responsive targeting peptide platelet liposomes were added to another dish. After incubation in the incubator for 1 hour, the samples were taken out and observed under a confocal microscope (scale bar: 10 μm).

[0229] Results Figure 6 It can be observed that the fluorescence of the peptide labeling in the first dish of cells is distributed around the cells, while the fluorescence of the peptide labeling in the second dish of cells enters the interior of the cells after adding the responsive targeted peptide platelet liposomes after fusion, indicating that the peptide and liposomes are successfully combined.

[0230] 7. Analysis of RSPNP Platelet Membrane Protein Components

[0231] The membrane proteins on platelets are the key components of the targeting of platelet liposomes. The platelet vesicles, platelet liposomes (PNP), targeted peptide platelet liposomes (RPNP) and responsive targeted peptide platelet liposomes (RSPNP) extracted in step 1 were subjected to immunoblotting analysis to detect the functional proteins on the platelets. The nanomaterials synthesized in step 2 were freeze-dried, added to the lysate to extract proteins, and immunoblotting was performed to analyze the platelet proteins on the surface of the nanomaterials.

[0232] like Figure 7 As shown, the nanomedicine synthesized by this method has protein bands similar to those of platelets on the surface, and has platelet-specific CD47, CD41 and CD36 proteins, which indicates that RSPNP-LycoD has functions similar to those of platelet membranes.

[0233] 8. Uptake of RSPNP nanomaterials by neurons and brain microvascular endothelial cells (microscope observation).

[0234] Platelet-derived liposomes (PNP), platelet-derived peptide liposomes (RPNP), and responsive platelet-derived peptide liposomes (RSPNP) were fluorescently labeled and added to the neuronal cell line (left) and the brain microvascular endothelial cell line (right) for 4 hours. Another dish of neuronal and brain microvascular endothelial cell culture dishes were taken, and after pretreatment with 10μM RVG29 peptide for 1 hour, an equal amount of fluorescently labeled RSPNP was added. After incubation, DAPI staining was performed, and the cells were observed under a microscope, and the fluorescence values ​​were counted (scale bar: 40μm).

[0235] Results Figure 8 It can be seen that both neurons and brain microvascular endothelial cells are more inclined to take up RSPNP-LycoD nanomaterials, and the uptake will be inhibited by peptide pretreatment.

[0236] 9. RSPNP targets ischemic injury areas in mice

[0237] In order to verify whether RSPNP can cross the blood-brain barrier and target the ischemic area, platelet-derived liposomes (PNP), platelet-derived peptide liposomes (RPNP) and responsive platelet-derived peptide liposomes (RSPNP) were labeled with fluorescent drugs respectively. Phosphate buffered saline (PBS) group and free fluorescent dye were used as controls. The same amount of fluorescence was injected into the ischemia-reperfusion model mice through the tail vein. The samples were collected 18 hours later and the drug distribution was observed by small animal in vivo imaging.

[0238] The results are as follows Fig.10 As shown, RSPNP was greatly enriched in the ischemic area and was significantly higher than that in other groups, proving that RSPNP has a strong ability to target the ischemic area.

[0239] 10. Experiment on the effect of RSPNP-LycoD on I / R-induced brain injury in mice

[0240] The free lycopene and deferoxamine (Free-LycoD) prepared in step 3 of Example 4 and the lycopene and deferoxamine-responsive targeted peptide platelet liposome (RSPNP-LycoD) nanomedicines prepared in Example 1-3, as well as a blank control phosphate buffer (PBS), were used to treat ischemic stroke model mice, and the cerebral infarction volume was detected by TTC staining after 24 hours.

[0241] The results are as follows Fig. 9 As shown in the figure, TTC dye can dye normal brain tissue red and infarcted tissue white. Compared with other treatment groups, RSPNP-LycoD significantly reduced the infarct area, indicating that RSPNP-LycoD has a significant neuroprotective effect on cerebral ischemia-reperfusion injury.

[0242] Example 6

[0243] The therapeutic effect of RSPNP-LycoD on the injury after thrombolysis in ischemic stroke

[0244] Therapeutic effects of RSPNP-LycoD in treating mice with cerebral infarction

[0245] (1) RSPNP-LycoD was prepared according to the method in Example 1.

[0246] (2) The mice were subjected to cerebral ischemia for 60 min by middle cerebral artery occlusion (MCAO). The occlusion was then removed and blood was reperfused for 24 h to establish an I / R model in mice, simulating the blood reperfusion after thrombolysis in human ischemic stroke.

[0247] (3) Four groups of preparations were set up, including PBS, Free-LycoD, RSPNP-Lyco and RSPNP-LycoD. During cerebral ischemia-reperfusion, each group of drugs was injected through the tail vein at a relative dose of 25 mg / kg body weight.

[0248] (4) After 24 hours of reperfusion, the mice were anesthetized with 5% chloral hydrate, the brains were removed by decapitation, and the brain slices were placed in 2% TTC staining solution in a 37°C constant temperature incubator in the dark for 7 minutes.

[0249] (5) Take photos and calculate the infarct volume according to Formula VI.

[0250] Percentage of infarct volume = (contralateral hemisphere volume - ipsilateral hemisphere non-infarct area volume) / contralateral hemisphere volume of infarcted area × 100% Formula VI.

[0251] The brain infarction volumes of mice in the PBS, Free, RSPNP-Lyco and RSPNP-LycoD groups were 24.77%, 27.61%, 8.71% and 6.5%, respectively. Compared with the other three groups, RSPNP-LycoD treatment reduced the infarction volume by 3.81 times (see Fig.10 ).

[0252] Example 7

[0253] RSPNP-LycoD treatment reduced the infarct volume of mice by 9.04-fold.

[0254] (1) RSPNP-LycoD was prepared according to the method in Example 1.

[0255] (2) The mice were subjected to cerebral ischemia for 60 min by middle cerebral artery occlusion (MCAO). The occlusion was then removed and blood was reperfused for 24 h to establish an I / R model in mice, simulating the blood reperfusion after thrombolysis in human ischemic stroke.

[0256] (3) Four groups of preparations were set up, including PBS, Free-LycoD, RSPNP-Lyco and RSPNP-LycoD. During cerebral ischemia-reperfusion, each group of drugs was injected through the tail vein at a relative dose of 25 mg / kg body weight.

[0257] (4) After 24 hours of reperfusion, the mice were anesthetized with 5% chloral hydrate, the brains were removed by decapitation, and the brain slices were placed in 2% TTC staining solution in a 37°C constant temperature incubator in the dark for 7 minutes.

[0258] (5) Take photos and calculate the infarct volume. The percentage of infarct volume = (contralateral hemisphere volume - ipsilateral hemisphere non-infarct area volume) / contralateral infarct area hemisphere volume × 100%. The brain infarct volumes of mice in the PBS, Free, RSPNP-Lyco and RSPNP-LycoD groups were 38.97%, 17.56%, 7.68% and 4.31%, respectively. Compared with the other three groups, RSPNP-LycoD treatment reduced the infarct volume by 9.04 times.

[0259] Example 8

[0260] The therapeutic effect of RSPNP-LycoD on mice with cerebral infarction

[0261] (1) RSPNP-LycoD was prepared according to the method in Example 1.

[0262] (2) The mice were subjected to cerebral ischemia for 60 min by middle cerebral artery occlusion (MCAO). The occlusion was then removed and blood was reperfused for 24 h to establish an I / R model in mice, simulating the blood reperfusion after thrombolysis in human ischemic stroke.

[0263] (3) Four groups of preparations were set up, including PBS, Free-LycoD, RSPNP-Lyco and RSPNP-LycoD. During cerebral ischemia-reperfusion, each group of drugs was injected through the tail vein at a relative dose of 25 mg / kg body weight.

[0264] (4) After 24 hours of reperfusion, the mice were anesthetized with 5% chloral hydrate, the brains were removed by decapitation, and the brain slices were placed in 2% TTC staining solution in a 37°C constant temperature incubator in the dark for 7 minutes.

[0265] (5) Take photos and calculate the infarct volume

[0266] The cerebral infarction volumes of mice in the PBS, Free, RSPNP-Lyco and RSPNP-LycoD groups were 23.37%, 13.15%, 9.73% and 9.31%, respectively. Compared with the other three groups, RSPNP-LycoD treatment reduced the infarction volume by 2.51 times.

[0267] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ROS-responsive platelet liposome nanoparticle, characterized in that: comprising a bilayer and deferoxamine wrapped by the bilayer; Lycopene is embedded in the middle of the bilayer, and the outer surface of the bilayer is modified with RVG29 polypeptide; The bilayer includes liposomes or is obtained by combining liposomes and platelet membranes; The liposome is prepared from lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000.

2. The ROS-responsive platelet liposome nanoparticles according to claim 1, characterized in that: The RVG29 polypeptide is linked to the maleimide bond of DSPE-PEG2000-MAL in the liposome via cysteine.

3. The ROS-responsive platelet liposome nanoparticles according to claim 1, characterized in that: The mass ratio of the RVG29 polypeptide to DSPE-PEG2000-MAL is 1:

1.

4. The ROS-responsive platelet liposome nanoparticles according to claim 1, characterized in that: The mass ratio of the lecithin, cholesterol, DSPE-PEG2000-MAL and DSPE-Se-Se-PEG2000 is 14:2:1:

1.

5. The ROS-responsive platelet liposome nanoparticles according to claim 1, characterized in that: When the bilayer is obtained by compounding the liposome and the platelet membrane, 1×10 8 ~2×10 8 Platelet membrane.

6. The ROS-responsive platelet liposome nanoparticle according to any one of claims 1 to 5, characterized in that: The concentration of lycopene is 0.7-1.5 mg / mL; The concentration of the deferoxamine is 2-5 mg / mL.

7. The method for preparing ROS-responsive platelet liposome nanoparticles according to any one of claims 1 to 6, characterized in that: The following steps are involved: Phosphatidylcholine, cholesterol, DSPE-PEG2000-MAL, DSPE-Se-Se-PEG2000, lycopene and deferoxamine are mixed to obtain liposomes loaded with lycopene and deferoxamine; Under the protection of inert gas, the lycopene and deferoxamine loaded liposomes and the cysteine-modified RVG29 polypeptide are subjected to a connection reaction to obtain lycopene and deferoxamine loaded liposomes with the surface modified with RVG29 polypeptide, which are denoted as RSNP-LycoD; When the bilayer also includes a platelet membrane, the method further includes mixing the lycopene and deferoxamine loaded liposomes modified with RVG29 polypeptide on the surface with platelets, and co-extruding them to obtain ROS responsive platelet liposome nanoparticles, RSPNP-LycoD nanoparticles.

8. Use of the ROS-responsive platelet liposome nanoparticles according to any one of claims 1 to 5 or the ROS-responsive platelet liposome nanoparticles prepared by the preparation method according to claim 7 in the preparation of drugs for preventing and / or treating nerve damage caused by iron overload and redox imbalance.

9. The use according to claim 8, characterized in that: The nerve damage caused by iron overload and redox imbalance includes nerve damage caused by reperfusion after thrombolysis or surgical thrombectomy in ischemic stroke.

10. The use according to claim 8 or 9, characterized in that: The dosage form of the drug includes at least one of the following: intravenous injection, capsule, oral solution, nasal spray and pill.

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