Pharmaceutical composition formed by combined drug loading of soft and hard nanoparticles and application of pharmaceutical composition

Abstract

The invention discloses a pharmaceutical composition formed by soft and hard nanoparticle combined drug loading and application of the pharmaceutical composition, and belongs to the field of biological medicine. According to the pharmaceutical composition disclosed by the invention, through co-incubation of two nanoparticles with different stiffness properties carrying drugs, the preparation method is simple. According to the pharmaceutical composition disclosed by the invention, the soft nanoparticles and the hard nanoparticles play a synergistic treatment role in lesion brain region and peripheral inflammation respectively, accumulation of target drug nanoparticles in lesion brain can be improved, and the pharmaceutical composition has a good treatment effect. According to the pharmaceutical composition, a novel pharmaceutical composition platform can be provided, particles with different rigidities are selected for collaborative delivery according to application scenes of different brain disease models, one-time pharmaceutical composition administration is achieved, multiple treatment and protection effects are achieved, and the treatment effect is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a drug composition formed by a combination of soft and hard nanoparticles for loading drugs and its application. Background Art

[0002] Stroke is the leading cause of long-term disability globally and the second leading cause of death worldwide, accounting for nearly 10% of the global mortality rate. Approximately 87% of stroke patients suffer from ischemic stroke, imposing a huge medical and economic burden on both patients and society. Ischemic stroke is mainly caused by cerebrovascular embolism or stenosis. During the acute phase, the brain tissue has insufficient local oxygen and glucose supply due to interrupted blood flow, resulting in blocked energy metabolism. Subsequently, brain damage in the area surrounding the infarction (ischemic penumbra) is caused by a series of secondary events, such as excitotoxicity, oxidative stress, and mitochondrial dysfunction. Irreversible damage occurs in the core infarction area of the brain tissue, and the death of nerve cells leads to the subsequent release of damage-associated molecular patterns (DAMPs), which in turn recruit peripheral immune cells. Neutrophils are the first blood-derived immune cells to invade the ischemic tissue, followed by monocytes. Studies have shown that a large influx of neutrophils into the brain exacerbates brain damage in ischemic stroke. After a stroke, neutrophils migrate through the endothelial blood vessel wall due to chemotactic factor attraction. Once in the brain, they release proteases such as pro-inflammatory factors, matrix metalloproteinases (MMPs), cathepsin G, reactive oxygen and nitrogen species, and inflammatory IL-1β. These substances damage the vascular endothelium and exacerbate the permeability of the blood-brain barrier (BBB) and brain edema. BBB damage leads to more peripheral neutrophils entering the damaged area. In addition, infiltrating neutrophils further exacerbate ischemic injury by forming extracellular traps decorated with proteases and cytotoxic histones and releasing substances such as neutrophil elastase (NE) and myeloperoxidase (MPO).

[0003] Existing treatment methods mainly aim to restore blood supply to the ischemic area, such as intravenous injection of tissue plasminogen activator (tPA) and mechanical thrombectomy to achieve vascular recanalization and thus restore blood flow. Blood reperfusion may generate excessive reactive oxygen species that damage cells, cause massive infiltration of peripherally activated immune cells, further exacerbate brain tissue damage and blood-brain barrier disruption, lead to increased brain edema, and may even result in persistent neurological dysfunction. Therefore, it is highly necessary to develop new treatment methods to alleviate brain tissue damage and neurological dysfunction caused by massive infiltration of peripheral immune cells.

[0004] In recent years, researchers have expected to use neuroprotective drugs such as antioxidants and anti-inflammatory drugs to salvage ischemic stroke injury. However, in addition to cell damage in the lesion area, ischemic stroke also activates the body's immune response, especially the innate immune system. In the acute inflammatory response, peripheral immune cells help the body resist infection or repair damaged tissues by increasing phagocytosis and clearance functions. This also leads to a faster rate of phagocytosis of exogenous particles, and after administration, the drug is taken up and cleared by a large number of activated immune cells in the periphery and cannot reach the lesion area to exert its effect. In addition, the tight junction proteins and adherens junction proteins of the blood-brain barrier form a physical seal between blood vessels and the brain parenchyma, which is another factor hindering drug entry into the brain. The existence of the blood-brain barrier makes it difficult for drug molecules with larger particle sizes (>100 nm), larger molecular weights (>500 Da), low lipid solubility or water solubility to penetrate the blood-brain barrier, resulting in extremely low drug bioavailability and the inability of the brain drug concentration to reach the therapeutic dose, limiting the efficacy. Therefore, it is urgent to select a suitable drug delivery method to help the drug exert its specific efficacy.

[0005] With the continuous development of nanotechnology, nanodrugs show great potential for meeting the treatment needs of brain diseases. The properties of nanoparticles such as particle size, zeta potential, and stiffness are key factors affecting their in vivo distribution and drug delivery efficiency. Summary of the Invention

[0006] Aiming at the deficiencies in the field of drug delivery treatment for current brain diseases, the present invention provides a drug composition in which dual-stiffness nanoparticles carry therapeutic drugs to exert dual therapeutic effects on the periphery and brain tissue respectively.

[0007] The present invention points out that within the same time, the efficiency of peripheral immune cells taking up hard nanoparticles is significantly higher than that of taking up soft nanoparticles. When particles with two stiffnesses, soft and hard, are administered together, the uptake rate and efficiency of peripheral immune cells for hard nanoparticles are faster and higher, while the uptake of soft nanoparticles will decrease. In the uptake by nerve cells, the efficiency of taking up soft nanoparticles is significantly higher than that of taking up hard nanoparticles.

[0008] The present invention utilizes the behavioral differences of nanoparticles with different stiffnesses at the cellular level to invent a drug combination in which nanoparticles with different stiffnesses carry therapeutic drugs to exert a synergistic effect for treating reperfusion injury after ischemic stroke. Hard nanoparticles are used to carry anti-inflammatory drugs (such as dexamethasone, methylprednisolone, diclofenac, piceatannol, indomethacin, aspirin) to inhibit the signal pathways related to immune cells and reduce the activation and migration of immune granulocytes under inflammatory conditions. Soft nanoparticles are used to carry neuroprotective agents (such as edaravone, nimodipine, carbamazepine, imipramine, butylphthalide) to regulate apoptosis and oxidative stress of nerve cells and reduce brain tissue damage after ischemic stroke. The present invention uses the two as a drug composition and administers it by intravenous injection, enabling a large number of immune cells in the periphery to uptake the hard nanoparticles, release anti-inflammatory drugs, and inhibit the infiltration of peripheral immune cells, especially neutrophils, into the brain during reperfusion of ischemic stroke, thereby reducing further inflammatory damage to brain tissue; the proportion of soft nanoparticles cleared by the peripheral immune system under this condition is reduced, increasing the concentration of neuroprotective agents in the brain and better exerting the neuroprotective effect. The present invention utilizes the differences in the in vivo fates of particles with different stiffnesses to prepare this drug composition, achieving a synergistic therapeutic effect at different times and in different spaces with a single administration.

[0009] The present invention provides the following technical solutions:

[0010] The present invention provides a novel drug composition for the treatment of ischemic stroke, enabling the hard and soft nanoparticles to carry drugs and exert a synergistic effect in the periphery and the lesion area respectively.

[0011] A drug composition formed by a combination of hard and soft nanoparticles carrying drugs, comprising:

[0012] Soft nanoparticles carrying neuroprotective drugs, wherein the material of the soft nanoparticles is selected from one or more of liposomes, phospholipids, vesicles, cell membranes, exosomes;

[0013] Hard nanoparticles carrying anti-inflammatory drugs, wherein the material of the hard nanoparticles is selected from one or more of poly(lactic-co-glycolic acid) (PLGA), silicon dioxide (SiO 2 )), gold nanoparticles, silver nanoparticles;

[0014] Wherein, the concentration ratio of the soft nanoparticles to the hard nanoparticles is 1:0.5 - 2.

[0015] Preferably, the neuroprotective drugs are selected from one or more of butylphthalide, edaravone, nimodipine, carbamazepine, imipramine, but are not limited thereto.

[0016] Preferably, the anti-inflammatory drugs are selected from one or more of piceatannol, dexamethasone, methylprednisolone, diclofenac, indomethacin, aspirin, but are not limited thereto.

[0017] Preferably, the mass ratio of the drug to the soft nanoparticles is 1:0.5 - 4. If the ratio is too large or too small, the soft nanoparticles cannot encapsulate the drug well.

[0018] The mass ratio of the drug to the hard nanoparticles is 1:1 - 3. Using the supercritical fluid technology or the microfluidic method, the hard nanoparticles are wrapped with a liposome membrane to unify the surface properties of the particles.

[0019] The brain-related diseases are ischemic stroke, traumatic brain injury, multiple sclerosis, Alzheimer's disease or Parkinson's disease.

[0020] The specific preparation methods of the present invention include the emulsion solvent evaporation method, the emulsion solvent diffusion method, the salting-out-emulsion diffusion method, the nanoparticle co-precipitation method, the supercritical fluid technology, the interfacial polymerization method, the hydrophilic polymer aggregation method and the emulsion polymerization method, etc.

[0021] The present invention also provides a preparation method of the drug composition, comprising the following steps:

[0022] (1) Prepare soft nanoparticles carrying a neuroprotective drug;

[0023] (2) Prepare hard nanoparticles carrying an anti-inflammatory drug;

[0024] (3) Co-incubate the soft nanoparticles and the hard nanoparticles at a predetermined concentration ratio to form the drug composition.

[0025] Preferably, the predetermined concentration ratio is: soft nanoparticles: hard nanoparticles is 1:0.5 - 2, and the co-incubation time is 0.5 - 2 hours.

[0026] The present invention also provides the application of the drug composition in the preparation of a drug for treating brain-related diseases.

[0027] Preferably, the brain-related diseases are ischemic stroke, traumatic brain injury, multiple sclerosis, Alzheimer's disease or Parkinson's disease.

[0028] In the embodiments of the present invention, ischemic stroke is taken as an example, but not limited thereto. The present invention designs two different stiffness nanoparticles, hard and soft, according to the actual disease model and application scenario. Because their uptake rates by immune cells and nerve cells are different, and their abilities to penetrate biological barriers are different, they play corresponding roles in the peripheral and brain tissues respectively.

[0029] The present invention also provides a drug delivery system, comprising the drug composition as described above, for simultaneously targeting the peripheral immune system and the brain tissue.

[0030] Specifically, the hard nanoparticles rapidly uptake and release anti-inflammatory drugs through peripheral immune cells, and the soft nanoparticles enter the brain tissue through the blood-brain barrier to release neuroprotective drugs.

[0031] Compared with the prior art, the beneficial effects of the pharmaceutical composition of the present invention are as follows:

[0032] (1) The pharmaceutical composition of the present invention is prepared by co-incubating drugs carried by two kinds of nanoparticles with different stiffness properties, and the preparation method is simple.

[0033] (2) The hard and soft nanoparticles of the pharmaceutical composition of the present invention play a synergistic therapeutic role in the diseased brain region and peripheral inflammation respectively, can improve the accumulation of target drug nanoparticles in the diseased brain, and have good therapeutic effects.

[0034] (3) The pharmaceutical composition of the present invention can provide a platform for a new type of pharmaceutical composition. According to the application scenarios of different brain disease models, particles with different stiffness are selected for synergistic delivery, so as to achieve a single administration of the pharmaceutical composition and exert multiple therapeutic and protective effects, greatly improving the therapeutic effect. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the present invention.

[0036] Figure 2 It is the surface morphology of nanoparticles with different stiffness in the present invention. Observation under a confocal microscope shows the co-localization of the membrane and nanoparticles.

[0037] Figure 3 It is the particle size and zeta potential of nanoparticles with different stiffness in the present invention.

[0038] Figure 4 It is the Young's modulus and 3D morphology of nanoparticles with different stiffness measured by atomic force microscopy in the present invention.

[0039] Figure 5 It is the uptake of nanoparticles with different stiffness at different time points in Raw264.7 cells in the present invention; wherein, **** indicates p < 0.0001.

[0040] Figure 6 It is the penetration of nanoparticles with different stiffness in an in vitro penetration model using a carrier composition of nanoparticles with different stiffness in the present invention; wherein, * indicates p < 0.05, and ** indicates p < 0.01.

[0041] Figure 7 It is the investigation of the biosafety of the pharmaceutical composition with different concentrations in vitro in the present invention.

[0042] Figure 8This shows the in vivo distribution of the pharmaceutical composition of the present invention in an ischemic stroke model. Comparison shows that the pharmaceutical composition is conducive to significantly increasing the amount of soft nanoparticles entering the brain; where, ** indicates p < 0.01.

[0043] Figure 9 This shows the in vivo distribution of the pharmaceutical composition of the present invention in an ischemic stroke model. Comparison shows that the hard nanoparticles in the pharmaceutical composition are more likely to be taken up and cleared peripherally and enter the liver; where, * indicates p < 0.05 and ** indicates p < 0.01.

[0044] Figure 10 This shows the treatment of the ischemic stroke with the pharmaceutical composition of the present invention, including the survival rate curve and the body weight change curve.

[0045] Figure 11 This shows the treatment of the ischemic stroke with the pharmaceutical composition of the present invention, including the recovery of the beam balance ability and the change of the neurological score during the 28-day treatment.

[0046] Figure 12 This shows the levels of inflammatory factors in the lesioned brain on the 1st day and the 3rd day after the treatment of the ischemic stroke with the pharmaceutical composition of the present invention; where, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0047] Figure 13 This shows the results of immunofluorescence staining and Nissl staining in the mouse brain tissue on the 28th day after the treatment of the ischemic stroke with the pharmaceutical composition of the present invention; where, bar = 20 μm. Detailed implementation mode

[0048] The schematic diagram of the present invention is as Figure 1 shown. The technical solution of the present invention will be described below with specific examples.

[0049] Example 1 Preparation and characterization of nanoparticles with different stiffness

[0050] Anionic liposomes (Liposome) were prepared by the thin film ultrasonic method. 1 mg of phosphatidylserine, 20 mg of soybean lecithin, and 2.5 mg of cholesterol were mixed and dissolved in 15 mL of chloroform. The chloroform was removed by rotary evaporation under reduced pressure, and then dried in a vacuum dryer for 2 h to further remove chloroform. After drying, 5 mL of dispersion medium was added to swell and hydrate the film, and it was placed in an ultrasonic cleaner for water bath sonication for 15 min. Then it was transferred to an EP tube for probe sonication for 3 min, with an ultrasonic power of 20 W, an ultrasonic time of 3 min (working for 2 s and resting for 3 s), and the number of ultrasonic times was 2 times. The preparation was completed.

[0051] Preparation of Lipo-PLGA. First, prepare PLGA nanoparticles. Oil phase: Dissolve 80 mg of PLGA in acetone solution and sonicate for dissolution. Aqueous phase: Heat 20 mL of ultrapure water on a magnetic stirrer, add polyvinyl alcohol (PVA) while stirring, and stir for about 20 min. After complete dissolution, slowly add the oil phase to the aqueous phase, stir for about 5 min, transfer to a eggplant-shaped flask, and under the conditions of 45 °C and a reduced pressure of 0.08 MPa, perform rotary evaporation under reduced pressure for 1.5 h to evaporate the organic solvent completely, obtaining a suspension of PLGA nanoparticles. After passing the suspension through a 0.8 μm filter membrane, use microfluidic technology to coat a lipid membrane on the surface of PLGA. The mass ratio of PLGA to phospholipid is 4:1, the injection volume ratio of the aqueous phase to the organic phase is 1:1, and the flow rate is 12 mL / min. Stir overnight to remove the organic reagent to complete the preparation of Lipo-PLGA.

[0052] Use laser confocal microscopy to observe whether Lipo is well-coated on the surface of PLGA to form a core-shell structure. Stain and label PLGA and the outer lipid membrane respectively, and use laser confocal microscopy to characterize the co-localization situation to further illustrate the structure of Lipo-PLGA. Seed Raw264.7 cells at 200,000 cells / well in a confocal dish, add 1 mL of culture medium, and culture overnight. Stain PLGA with DiI (1:500), and dissolve the liposome with DiD in ethanol (1:500). Administer the carrier at a dose of 0.5 mg / mL and 100 μL. After 4 h of administration, fix the cells, stain with DAPI, and take pictures using laser confocal microscopy. The results are as Figure 2 . The experimental results show strong co-localization of DiI and DiD signals, and the Pearson coefficient is 0.89 ± 0.05, indicating that the lipid is successfully coated on the surface of PLGA to form Lipo-PLGA nanoparticles.

[0053] Use a Malvern particle size and zeta potential analyzer (DLS) to measure the particle size and zeta potential of the particles, and use a transmission electron microscope (TEM) to observe the particle morphology. The results are as Figure 3 . The experimental results show that Liposome and Lipo-PLGA have similar particle sizes and zeta potentials.

[0054] Example 2 Mechanical Properties of Nanoparticles with Different Stiffness

[0055] Use an atomic force microscope to measure the Young's modulus and 3D topography of Liposome and Lipo-PLGA nanoparticles respectively. Fix the well-dispersed liquid on a glass slide after air-drying. According to the test content, select the contact mode, install the probe, adjust the photodetector, and detect and save the original file, 2D and 3D topography, and Young's modulus. The experimental results are as Figure 4In summary, nanoparticles with similar particle sizes, potentials, similar surface conditions but significantly different Young's moduli were prepared by this preparation method. Therefore, Liposome was selected as Soft NPs and Lipo-PLGA as Stiff NPs for subsequent applications.

[0056] Example 3 Uptake of Nanoparticles with Different Stiffness by Cells

[0057] Prepare Soft NPs (1 mg / mL) and Stiff NPs (1 mg / mL) with similar surface properties, and label Soft NPs (1 mg / mL, 5 mL) and Stiff NPs (1 mg / mL, 5 mL) with an equal amount of DiD (1:1000) dye. Seed Raw264.7 cells at 200,000 cells / well in a 12-well plate and administer 100 μL of the drug the next day. Use flow cytometry to observe the average fluorescence intensity in cells 1 h, 2 h, 4 h, and 6 h after drug administration. The results are as Figure 5 , under the condition of keeping other conditions the same, Stiff NPs with greater stiffness were significantly more taken up by Raw264.7 cells at each time point. This example of the present invention proves the conclusion that nanoparticles with greater stiffness are easily taken up by immune cells.

[0058] Example 4 Penetration of Carrier Compositions Composed of Nanoparticles with Different Stiffness in an in vitro Penetration Model

[0059] Mix the nanoparticles with different stiffness prepared in the examples according to a mass ratio of 1:1 respectively to form carrier compositions of nanoparticles with different stiffness, and conduct penetration experiments. Investigate whether combined administration can enable more hard nanoparticles to be taken up by neutrophils, thereby protecting soft nanoparticles from being taken up by immune cells and penetrating more into the lower layer and being taken up by nerve cells PC12.

[0060] Specific experimental design: Extract primary neutrophils from ICR mice in vitro, add 2×10 5 , 200 μL of neutrophils in the upper layer, and plate 10 5 , 1 mL of PC12 cells in the lower layer in advance. Add the sample to the upper layer and investigate the particle uptake by PC12 cells penetrating into the lower layer and the particle phagocytosis by neutrophils in the upper layer 3 h later. The experimental groups are as follows:

[0061] StiffNPs (1 mg / mL, 10 μL, stained with DiD 1:400);

[0062] Combined administration of StiffNPs (1 mg / mL, 10 μL, stained with DiD 1∶400) + Soft NPs (1 mg / mL, 10 μL);

[0063] Soft NPs (1 mg / mL, 10 μL, stained with DiD 1:400);

[0064] Combined administration of Stiff NPs (1 mg / mL, 10 μL) and Soft NPs (1 mg / mL, 10 μL, stained with DiD 1:400).

[0065] The results are as Figure 6 , indicating that the carrier composition of the present invention will significantly reduce the percentage of cellular uptake of Soft NPs by upper-layer neutrophils. From the investigation of the average fluorescence intensity, it can be found that the carrier composition of the present invention will significantly increase the amount of Stiff NPs taken up by neutrophils and reduce the amount of Soft NPs taken up by neutrophils. The ability of PC12 cells to take up Soft NPs is significantly stronger than that of Stiff NPs. Moreover, after the particles penetrate into the lower layer, the pharmaceutical composition of the present invention will significantly enhance the percentage of cellular uptake of Soft NPs by PC12 cells. This example proves in vitro that the carrier composition of the present invention can increase the uptake of Stiff NPs by immune cells, while more Soft NPs penetrate into the lower layer after evading uptake and are taken up by the neuronal cell line PC12 cells.

[0066] Example 5 Safety of the Pharmaceutical Composition Formed by the Carrier Composition Composed of Nanoparticles with Different Stiffness Carrying Drugs

[0067] Based on the differences in the cellular uptake behaviors of nanoparticles with different stiffness in the previous foundation, the present invention carries an anti-inflammatory drug in Stiff NPs and a neuroprotective drug in Soft NPs, which play their respective roles to form the pharmaceutical composition of the present invention.

[0068] Prepare Pic@Stiff NPs. Oil phase: Dissolve 80 mg of PLGA and piceatannol (Pic) in an acetone solution, and the mass ratio of Pic to PLGA is 1:20, and dissolve by ultrasonic wave. Aqueous phase: Heat 20 mL of ultrapure water on a magnetic stirrer, add PVA while stirring, and stir for about 20 min. After complete dissolution, slowly add the oil phase to the aqueous phase, stir for about 5 min, transfer to an eggplant-shaped flask, and under the conditions of 45 °C and a reduced pressure of 0.08 MPa, evaporate the organic solvent by rotary evaporation under reduced pressure for 1.5 h to obtain a suspension of PLGA nanoparticles. After passing the suspension through a 0.8-μm filter membrane, the preparation is completed. Use the microfluidic method to coat the surface of Pic@PLGA to form Pic@Stiff NPs.

[0069] Prepare NBP@Soft NPs. According to Example 1, add n-butylphthalide (NBP) to it. Generally, it is considered that the mass ratio of NBP to liposome can be 3:5. Form NBP@Soft NPs.

[0070] Incubate Pic@StiffNPs and NBP@Soft NPs in a shaker (37 °C, 50 rpm) for 10 min at a ratio of 1:1 to prepare the pharmaceutical composition of the present invention.

[0071] To characterize whether the pharmaceutical composition is toxic to normal cells, using Raw264.7 as a model cell, the biosafety of the pharmaceutical composition at different concentrations (calculated based on the concentration of the hard nanoparticles Pic@Stiff NPs) was investigated by CCK8 assay. The results are as Figure 7 , indicating that the cell viability of Raw264.7 cells will not be greatly affected after administration of the pharmaceutical composition of the present invention at different concentrations for 12 h, and the cell survival rate of the experimental groups is all above 80%.

[0072] In vivo distribution of the pharmaceutical composition in Example 6 in an ischemic stroke disease model

[0073] Establish an ischemic stroke model. After anesthetizing the mice with 1% sodium pentobarbital, fix them in the supine position. Make an incision in the neck, separate the muscles and glands, and find the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) on the left side. Ligate the proximal end of the CCA, make a slipknot at the distal end, and clamp the upper part of the slipknot with a hemostatic clip. Make an incision above the ligation site of the CCA with scissors, insert a suture with a diameter of 0.23 ± 0.01 mm at the head from the ECA into the ICA to the hemostatic clip, tie the slipknot tightly and then remove the hemostatic clip, and continue to push the suture until obvious resistance appears and then stop to block the blood flow of the middle cerebral artery (MCA). Then suture the wound and transfer the mice to an incubator at a constant temperature of 37 °C to maintain body temperature. After 1 h, pull out the suture to restore collateral circulation blood supply, thus entering the reperfusion stage.

[0074] After modeling, score and administer drugs to the mice. To better illustrate the in vivo distribution, the hard and soft nanoparticles are labeled and then administered. The experimental groups are as follows:

[0075] NBP@Soft NPs: Inject 100 μL of 1000 μg / mL NBP@Soft NPs (DiD-labeled, 1:200) alone;

[0076] Pic@StiffNPs + NBP@Soft NPs(Dye): Mix 100 μL of 1000 μg / mL unlabeled Pic@StiffNPs and 100 μL of 1000 μg / mL NBP@Soft NPs (DiD-labeled, 1:200), and inject 200 μL;

[0077] Pic@Stiff NPs: Inject 100 μL of 1000 μg / mL Pic@Stiff NPs (DiD-labeled, 1:200) alone;

[0078] Pic@Stiff NPs(Dye) + NBP@Soft NPs: Mix 100 μL of 1000 μg / mL Pic@Stiff NPs (DiD-labeled, 1:200) and 100 μL of unlabeled 1000 μg / mL NBP@Soft NPs, and inject 200 μL.

[0079] After 24 h of drug circulation in the body, perfuse the hearts of the mice, and take the heart, liver, spleen, lung, kidney, and brain tissues of the mice for in vivo distribution.

[0080] The results are as Figure 8 、 9 , indicating that the pharmaceutical composition of the present invention can increase the entry of soft nanoparticles carrying the neuroprotective drug NBP into the brain tissue and reduce the amount of soft nanoparticles cleared by immune cells in the periphery and entering the liver. Moreover, the hard nanoparticles carrying the anti-inflammatory drug are more taken up by immune cells in the periphery to play a role, with an increased amount entering the liver and a decreased amount entering the brain tissue. The pharmaceutical composition of the present invention realizes the synergistic effect of hard and soft nanoparticles.

[0081] Therapeutic ability of the pharmaceutical composition in an ischemic stroke disease model in Example 7

[0082] Establish an ischemic stroke model according to the method in Example 6. Use the pharmaceutical composition Mixed group (NBP@Soft NPs and Pic@Stiff NPs are mixed in a volume ratio of 1:1 and administered in combination, 10 mg / kg), NBP group (NBP@Soft NPs, 10 mg / kg), and NT group (non-treatment group) in the present invention. After modeling, perform scoring, grouping, and drug administration.

[0083] The results are as Figure 10 、 Figure 11 , indicating that the pharmaceutical composition of the present invention shows a better survival rate compared with the commonly used NBP in clinics and delays the death time of the first mouse; the pharmaceutical composition group greatly reduces the mortality rate of MCAO / R mice within 10 days. And from Figure 10As can be seen from the body weight curve, the body weight of the group administered with the pharmaceutical composition of the present invention showed an increasing trend on the third day after administration. From Figure 11 In the neurological score of Figure 11 , it can be seen that the mNSS showed a decreasing trend starting from the second day, mainly reflected in the recovery of balance function, motor function and sensory function. In contrast, the mNSS of the group administered alone showed a decreasing trend on the 4th day after administration, and the body weight began to increase slowly on the 4th and 5th days. In addition, all the mice with the highest scores at the beginning after the administration of the pharmaceutical composition of the present invention survived and the behavioral scores gradually decreased, and the behavioral scores on Day 7 were only 8 or 9 points. However, the mice with the highest scores in the other two groups all died, or there was no obvious improvement in the behavior.

[0084] The treatment experiment shows that the group administered with the pharmaceutical composition of the present invention has the therapeutic effects of improving the survival rate of MCAO / R mice, inhibiting the weight loss of mice earlier, and improving the behavior of mice earlier.

[0085] Ability of the pharmaceutical composition in Example 8 to inhibit the inflammatory level in an ischemic stroke disease model

[0086] The ischemic stroke model was established and grouped for administration according to the method in Example 7. At 24 h and the 3rd day after administration, the whole brain tissue was perfused and removed, cut into left (lesioned brain) and right (contralateral brain) hemispheres, and 3 times the volume of PBS (mL:g = 3:1) was added as a buffer solution, and ground in a pre-cooled automatic rapid grinder (60 Hz, 1 min, 2 times) to obtain 25% brain tissue homogenate. Centrifuge at 20,000 g and 4 °C for 10 min, take the supernatant for testing, and after diluting 5 times, operate according to the instructions of the mouse TNF-α and interleukin-1β (IL-1β) kits to measure the concentrations of TNF-α and IL-1β in the left hemisphere (lesioned brain) of the mouse. And, the supernatant of the brain tissue homogenate was diluted 2.5 times (2 times in the experiment on Day 3), the protein concentration in the left brain tissue was quantified by a BCA kit, and the levels of IL-1β and TNF-α in the brain tissue were calculated according to the protein content. The experimental results are as Figure 12 shown, indicating that the pharmaceutical composition of the present invention can inhibit inflammation earlier and faster at 24 h after administration, and this effect can still be seen until the 3rd day after administration. The group administered with the pharmaceutical composition of the present invention significantly inhibited the levels of TNF-α and IL-1β on the lesioned brain side. And this result also shows that because the pharmaceutical composition of the present invention can show a better inhibitory effect on the inflammatory level of the lesioned brain tissue at 24 h after administration, that is, it can inhibit inflammation and protect nerve cells faster in the early stage. Therefore, even on Day 3, when the inflammatory inhibition levels of the two treatment groups are similar, the combined administration group will still show better curative effects than the group administered with NBP alone in the long-term treatment experiment. That is, the pharmaceutical composition of the present invention can better exert the inflammatory inhibitory effect.

[0087] Neuroprotective ability of the pharmaceutical composition in an ischemic stroke disease model

[0088] The ischemic stroke model was established and grouped for drug administration according to the method in Example 7. After 28 days of the long-term treatment experiment, the brain tissue was taken out by perfusion intact, fixed with 4% paraformaldehyde and embedded in paraffin, and coronal sections with a thickness of 6 μm were prepared for easy observation of the cortex and hippocampal regions. Before staining, the sections were soaked according to the following steps for dewaxing: xylene for 15 min, absolute ethanol for 5 min, 90% ethanol for 2 min, and double-distilled water for 2 min.

[0089] Nissl staining: The sections were immersed in Nissl staining solution and stained in a water bath at 50 °C for 30 min. After washing twice with 70% ethanol, they were sealed with neutral resin. Nissl bodies are inherent components in the cytoplasm of neurons and appear dark blue after staining, while the cytoplasm appears light blue.

[0090] Immunofluorescence staining: The sections were permeabilized in 0.1% Triton (100×) for 15 min, washed, and then blocked with goat serum at room temperature for 1 h. Immunofluorescence staining was performed by incubating overnight at 4 °C with GFAP primary antibody (Abcam, ab68428, 1:1000). Then, goat anti-rabbit IgG FITC secondary antibody (Abcam, ab6717, 1:1000) was used, and stained with 10 μg / mL DAPI. GFAP is the main intermediate filament protein in mature astrocytes and is a cell-specific marker. The GFAP positive signal reflects the formation of astrocytic scars.

[0091] TdT-mediated dUTP nick end labeling (TUNEL) staining: The brain sections were immersed in TUNEL staining solution containing TAT and FITC-dUTP (1:9, W / V) and stained for 60 min. The TUNEL positive signal reflects neuronal apoptosis.

[0092] All brain tissue sections were scanned by a virtual digital slide scanning system. The experimental results are as Figure 13 . According to the results, in the NT group, the number of Nissl bodies in the infarcted area of the mouse brain tissue decreased significantly, and dissolution and fragmentation occurred, indicating extensive damage to nerve cells. After treatment with the pharmaceutical composition of the present invention, the number of Nissl bodies in the infarcted area of the mouse brain in the Mixed group increased and the structure tended to be complete, indicating the recovery of neuronal activity. Moreover, compared with the NBP group and the NT group, the astrocytic scars formed at the brain injury site in the Mixed group treated with the pharmaceutical composition of the present invention were less, and the apoptotic neuron signal was weaker. That is, the pharmaceutical composition of the present invention can better play a neuroprotective role.

Claims

1. A pharmaceutical composition formed by combining soft and hard nanoparticles to carry drugs, characterized in that: include: Soft nanoparticles carrying neuroprotective drugs, wherein the material of the soft nanoparticles is selected from one or more of liposomes, phospholipids, vesicles, cell membranes, and exosomes; Hard nanoparticles carrying anti-inflammatory drugs, wherein the material of the hard nanoparticles is selected from one or more of poly(lactic-co-glycolic acid), silicon dioxide, gold nanoparticles, and silver nanoparticles; Wherein, the concentration ratio of the soft nanoparticles to the hard nanoparticles is 1:0.5-2.

2. The pharmaceutical composition according to claim 1, characterized in that The neuroprotective drug is selected from one or more of butylphthalide, edaravone, nimodipine, carbamazepine, and imipramine.

3. The pharmaceutical composition according to claim 1, characterized in that The anti-inflammatory drug is selected from one or more of picrosan, dexamethasone, methylprednisolone, diclofenac, indomethacin and aspirin.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The mass ratio of the drug to the soft nanoparticles is 1:0.5-4; The mass ratio of the drug to the hard nanoparticles is 1:1-3.

5. A method for preparing the pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Preparation of soft nanoparticles carrying neuroprotective drugs; (2) Preparation of hard nanoparticles carrying anti-inflammatory drugs; (3) co-incubating the soft nanoparticles and the hard nanoparticles at a predetermined concentration ratio to form the pharmaceutical composition.

6. The preparation method according to claim 5, characterized in that: The predetermined concentration ratio is: soft nanoparticles: hard nanoparticles is 1:0.5-2, and the co-incubation time is 0.5-2 hours.

7. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating brain-related diseases.

8. The use according to claim 7, characterized in that: The brain-related disease is ischemic stroke, traumatic brain injury, multiple sclerosis, Alzheimer's disease or Parkinson's disease.

9. A drug delivery system, characterized in that: A pharmaceutical composition comprising any one of claims 1 to 4, for simultaneously targeting the peripheral immune system and brain tissue.

10. The drug delivery system according to claim 9, characterized in that The hard nanoparticles are rapidly taken up by peripheral immune cells to release anti-inflammatory drugs, and the soft nanoparticles pass through the blood-brain barrier and enter brain tissue to release neuroprotective drugs.

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