Mesenchymal stem cell membrane bionic nano-enzyme for targeted therapy of Parkinson's disease as well as preparation method and application of mesenchymal stem cell membrane bionic nano-enzyme

Abstract

The invention discloses a mesenchymal stem cell membrane bionic nano-enzyme for targeted therapy of Parkinson's disease as well as a preparation method and application of the mesenchymal stem cell membrane bionic nano-enzyme. The mesenchymal stem cell membrane bionic nano enzyme comprises an inner core and a mesenchymal stem cell membrane, wherein the inner core is composed of mesoporous polydopamine nanoparticles, and the mesenchymal stem cell membrane coats the inner core. The medicine contains the mesenchymal stem cell membrane bionic nano enzyme. The mesenchymal stem cell membrane bionic nano-enzyme disclosed by the invention shows good biocompatibility, and can be homed to a Parkinson disease region in the brain under the mediation of CXCR4 and VLA-4 specific membrane proteins. In addition, through the CD47 membrane protein expressed on the surface, the removal of the immune system can be avoided, so that the action time in the body is prolonged. The nano-enzyme is helpful for eliminating reactive oxygen free radicals, reducing oxidative stress level and reducing damage of neuroinflammation to dopaminergic neurons, so that safe and efficient treatment of Parkinson's disease is realized.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular, to a mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease, and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is the second most prevalent neurodegenerative disease globally, with clinical manifestations of motor and cognitive dysfunction, affecting millions of elderly people worldwide, especially those aged 65 and above. The main pathological feature of Parkinson's disease is the degeneration of dopaminergic neurons in the substantia nigra striatum of the midbrain and the depletion of dopamine (DA). Currently, the clinical practice mainly adopts replacement therapy mainly based on supplementing dopamine, but long-term use will lead to a decline in efficacy and a large number of complications. Therefore, it is of great significance to explore new strategies for the treatment of Parkinson's disease.

[0003] The pathogenesis of Parkinson's disease is closely related to oxidative stress and neuroinflammation, which is characterized by the excessive production of reactive oxygen species (ROS). The increase in ROS levels will lead to oxidative stress and promote the abnormal accumulation of α-synuclein (α-syn), thereby inducing the polarization of microglia to the pro-inflammatory M1 phenotype. In addition, α-syn will also exacerbate the oxidative damage of dopaminergic neuron mitochondria, thereby further generating ROS. Therefore, scavenging ROS to combat neuroinflammation is a new method for the treatment of PD.

[0004] Currently, some small molecule drugs, natural enzymes, and inorganic nanozymes are used to scavenge ROS in inflammatory diseases. However, these drugs face huge challenges, including difficulty in crossing the blood-brain barrier, limited stability, and potential toxicity. As a natural biopolymer, polydopamine (PDA) has free radical scavenging and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT), thereby protecting the body from ROS-induced damage under inflammatory conditions. In addition, PDA also has characteristics such as small particle size, modifiability, and good biocompatibility, and has obvious advantages in the treatment of brain diseases. Although we advocate using PDA nanoparticles to treat Parkinson's disease (PD), due to the existence of the blood-brain barrier (BBB), a special structure in the brain, which blocks the entry of most therapeutic drugs into the brain, its insufficient targeting in the brain remains a huge challenge that needs attention.

[0005] Mesenchymal stem cells have the ability of self-renewal and multi-directional differentiation potential. Mesenchymal stem cells express a variety of membrane proteins on their surface, including CXCR4 (chemokine receptor), VLA-4 (integrin), and CD47. Very late activation antigen VLA-4 binds to vascular adhesion molecules on endothelial cells damaged by inflammation within the BBB, promoting their crossing of the BBB. Therefore, VLA-4 mediates the rolling, adhesion, and directional migration of mesenchymal stem cells through the vascular endothelial system towards the chemically damaged area. CD47 is a transmembrane glycoprotein that can interact with signal regulatory proteins on macrophages, inhibit phagocytosis, and prolong the circulation time of mesenchymal stem cells in vivo, thereby improving the therapeutic effect. The SDF-1 / CXCR4 signaling pathway can regulate the homing of mesenchymal stem cells in an oxidative stress environment. The elevated level of SDF-1 in the Parkinson's disease lesion area promotes the chemotaxis of mesenchymal stem cells, thereby enhancing their targeting ability to the affected area. Based on this, by coating the surface of nanoparticles with mesenchymal stem cell membranes to achieve a biomimetic effect, it can effectively help the nanoparticles enter the brain to play a therapeutic role.

[0006] Based on this, it is an urgent problem to be solved by the present invention to provide a targeted drug for Parkinson's disease that can effectively penetrate the blood-brain barrier, be targeted and delivered to the lesion area to scavenge ROS, reduce brain damage caused by neuroinflammation, and protect neurons. Summary of the Invention

[0007] Aiming at the above-mentioned prior art, the purpose of the present invention is to construct a mesenchymal stem cell membrane biomimetic nanoenzyme for targeted treatment of Parkinson's disease based on the excellent free radical scavenging effect and antioxidant enzyme-like properties of mesoporous polydopamine nanoparticles, as well as the great potential of the biomimetic effect of natural cell membranes in brain drug delivery.

[0008] Another object of the present invention is to provide a preparation method and application of the above-mentioned mesenchymal stem cell membrane biomimetic nanoenzyme for targeted treatment of Parkinson's disease.

[0009] To achieve the above object, the present invention provides a mesenchymal stem cell membrane biomimetic nanoenzyme for targeted treatment of Parkinson's disease, the mesenchymal stem cell membrane biomimetic nanoenzyme includes a core composed of mesoporous polydopamine nanoparticles, and a mesenchymal stem cell membrane coating the core. Among them, the mesoporous polydopamine nanoparticles have antioxidant enzyme effects, and the mesenchymal stem cell membrane coats the mesoporous polydopamine nanoparticles.

[0010] Preferably, the particle size of the mesenchymal stem cell membrane biomimetic nanoenzyme is 140 - 260 nm. That is, the particle size of the mesenchymal stem cell membrane biomimetic nanoenzyme is about 200 nm.

[0011] The present invention also provides a preparation method of the mesenchymal stem cell membrane biomimetic nanoenzyme according to the above, the preparation method includes:

[0012] S100. Preparation of mesoporous polydopamine nanoparticles: In the presence of an aqueous ethanol solution, dopamine hydrochloride and a surfactant are mixed, and then a templating agent is added thereto to obtain a nanoemulsion; ammonia water is added to the obtained nanoemulsion for a polymerization reaction to obtain a dendritic mesoporous polymer. After centrifugation, the precipitate is taken and washed to remove the excess surfactant and templating agent. After microfiltration, it is freeze-dried to obtain mesoporous polydopamine nanoparticles;

[0013] S200. Preparation of mesenchymal stem cell membrane biomimetic nanozyme: Under ice bath conditions, the mesoporous polydopamine nanoparticles obtained in step S100 and the mesenchymal stem cell membrane are mixed at a weight ratio of 40 - 60:1 and then ultrasonically oscillated. Then, the unencapsulated mesenchymal stem cell membrane is removed by centrifugation to obtain a mesenchymal stem cell membrane biomimetic nanozyme.

[0014] Preferably, in step S100, the surfactant is F - 127 surfactant;

[0015] and / or, the templating agent is tetramethylbenzene;

[0016] and / or, the weight ratio of the amount of dopamine hydrochloride to the surfactant is 1:1.5 - 3, the volume ratio of ethanol to water in the aqueous ethanol solution is 1:0.8 - 1.2, and the amount of the aqueous ethanol solution is 80 - 120 mL relative to 1 g of the surfactant;

[0017] and / or, the amount of the templating agent is 0.8 - 1.2 mL relative to 1 g of the surfactant.

[0018] Preferably, in step S100, the centrifugation speed during the centrifugation process is 15000 - 18000 r / min, and the centrifugation time is 12 - 18 min;

[0019] and / or, the washing process is to ultrasonically wash 3 times alternately with water and ethanol;

[0020] and / or, the filtration pore size of the microfiltration is 0.4 - 0.5 μm.

[0021] Preferably, in step S200, the mesenchymal stem cell membrane is a mesenchymal stem cell membrane with a particle size of 140 - 260 nm obtained by extrusion using a liposome extruder.

[0022] Preferably, the mesenchymal stem cell membrane is purified and amplified by the whole bone marrow adherent method and extracted by the repeated freeze - thaw method.

[0023] Preferably, the method for preparing the mesenchymal stem cell membrane specifically includes: collecting the bone marrow suspension of rats, inoculating it in MEM medium containing fetal bovine serum and penicillin-streptomycin, culturing it in a constant temperature incubator, changing the medium to remove other non-adherent cells after culturing for 42-54 hours, and continuing to culture the adherent MSC cells. After culturing for 4-6 passages, collect the cell-containing solution; first place the collected cell-containing solution in a -80°C refrigerator for 5 minutes, then place it in a 37°C shaker for 4-7 minutes, and perform the operation continuously for 3-5 times. Then, centrifuge at 2500g-3500g for 3-8 minutes to extract the supernatant, and then centrifuge the supernatant at 40000g-60000g for 25-40 minutes to obtain the precipitate, thus obtaining the mesenchymal stem cell membrane.

[0024] The following details the specific operation process for its preparation method:

[0025] Obtaining the mesenchymal stem cell membrane: Extract mesenchymal stem cells from the limb bones of 4-week-old healthy male SD rats, purify and amplify them using an optimized whole bone marrow adherent method, and extract the mesenchymal stem cell membrane using the repeated freeze-thaw method. Specifically, the operation process is as follows: Decapitate the rats and soak them in 75% ethanol. After 10 minutes, dissect the limbs in a ventilated ultra-clean workbench. Remove the muscle tissue with scissors, then isolate the femurs, tibias, etc. Cut open both ends of the bones with scissors and vertically place them into an EP tube with an open bottom to centrifuge and collect the mixture. Filter it through a 70μm cell filter membrane, collect the cell mixture, inoculate it in MEM medium containing 10% fetal bovine serum and 100IU / mL penicillin-streptomycin, and culture it in a constant temperature incubator at 37°C and 5% CO2. After 48 hours, change the medium to remove other non-adherent cells, and continue to culture the adherent MSC cells. After about 5 passages, collect all the cells for extracting the cell membrane. First place the cell-containing solution (i.e., the cells collected after culturing for 5 passages above) in a -80°C refrigerator for 5 minutes, then place it in a 37°C shaker for 5 minutes, and perform the operation continuously for 3 times (i.e., repeated freeze-thaw). Then, centrifuge at 3000g for 5 minutes to extract the supernatant, and then centrifuge the supernatant at 50000g for 30 minutes to obtain the precipitate, which is the mesenchymal stem cell membrane. Store it in a -80°C refrigerator for later use.

[0026] Obtaining mesoporous polydopamine nanoparticles: Dissolve 1.0 g of F-127 and 0.5 g of DA in 100 mL of a water and ethanol mixture with a volume ratio of 1:1, and vigorously stir at room temperature for 10 minutes to obtain a clear solution. Slowly inject 1.0 mL of TMB into the solution and stir at a constant speed of 500 rpm for 30 minutes to form a milky white F-127 / TMB nanoemulsion system. Then, gradually add 5.0 mL of concentrated ammonia water (NH4OH) dropwise to the above mixture to induce the self-polymerization of dopamine oligomers. After the addition is complete, continue the reaction for 2 hours. Centrifuge the formed dendritic mesoporous TMB / F127 / PDA polymer nanospheres at 16,500 r for 15 minutes for separation, and wash them 3 times with an ultrasonic probe using water and ethanol in sequence to remove the soft template. Finally, filter through a 0.45 μm microporous membrane and freeze-dry to obtain mesoporous polydopamine nanoparticles (mPDA) for standby.

[0027] Obtaining mesenchymal stem cell membrane biomimetic nanozymes: Use a liposome extruder to extrude the collected mesenchymal stem cell membranes (i.e., MSC cell membranes). Select polycarbonate membranes with pore sizes of 400 nm and 200 nm in sequence to prepare MSC cell membranes with a particle size of approximately 200 nm. Mix the synthesized mesoporous polydopamine nanoparticles with mesenchymal stem cell membranes at a ratio of 50:1 and ultrasonicate in an ice bath for 5 minutes to coat the mesoporous polydopamine nanoparticles with MSC cell membranes. Centrifuge at 16,500 r for 15 minutes to remove the unencapsulated MSC cell membranes, and obtain mesenchymal stem cell membrane-coated mesoporous polydopamine biomimetic nanozymes (i.e., mesenchymal stem cell membrane biomimetic nanozymes, denoted as mPDA@MSC).

[0028] The present invention also provides the application of the above-mentioned mesenchymal stem cell biomimetic nanozymes in the preparation of drugs for treating Parkinson's disease.

[0029] The present invention also provides a drug for targeted treatment of Parkinson's disease, which contains the above-mentioned mesenchymal stem cell membrane biomimetic nanozymes, or contains the mesenchymal stem cell membrane biomimetic nanozymes prepared by the above-mentioned preparation method.

[0030] In the present invention, various specific proteins on the mesenchymal stem cell membrane in mPDA@MSC can play the following roles: Mesenchymal stem cells express various membrane proteins on their surface, including CXCR4 (chemokine receptor), VLA-4 (integrin), and CD47. Very late activation antigen VLA-4 binds to vascular adhesion molecules on the inflamed endothelial cells within the BBB (blood-brain barrier), promoting their crossing of the BBB. Therefore, VLA-4 mediates the rolling, adhesion, and directional migration of mesenchymal stem cells through the vascular endothelial system towards the chemically damaged area. CD47 is a transmembrane glycoprotein that can interact with signal regulatory proteins on macrophages, inhibiting phagocytosis and prolonging the circulation time of mesenchymal stem cells in vivo, thereby enhancing the therapeutic effect. The SDF-1 / CXCR4 signaling pathway can regulate the homing of mesenchymal stem cells in an oxidative stress environment. The elevated level of SDF-1 in the Parkinson's disease lesion area promotes the chemotaxis of mesenchymal stem cells, thus enhancing their targeting ability to the affected area.

[0031] The mPDA@MSC of the present invention can effectively penetrate the blood-brain barrier. When it reaches the brain lesion site, by scavenging free radicals and exerting antioxidant enzyme activity, it significantly reduces ROS and alleviates neuroinflammation caused by oxidative stress, reducing damage to the brain. It can also protect dopaminergic neurons by regulating mitochondrial function and eliminate α-syn. In addition, it can inhibit the M1 polarization of microglia induced by the inflammatory environment and promote its transformation into the anti-inflammatory M2 phenotype, which may further reduce brain damage, thus achieving the therapeutic effect against Parkinson's disease.

[0032] Based on this, the present invention has the following advantages:

[0033] 1. In the present invention, due to its mesoporous structure, the mesoporous polydopamine nanoparticles have a larger specific surface area and can exert more excellent scavenging activity of reactive oxygen species and anti-peroxidase activity, effectively reducing brain damage and neuronal degeneration and necrosis caused by neuroinflammation in Parkinson's disease.

[0034] 2. In the present invention, there are various specific receptors on the mesenchymal stem cell membrane, including CXCR4, VLA-4, and CD47, which can effectively help the nanozyme penetrate the blood-brain barrier and reach the Parkinson's disease lesion site.

[0035] 3. In the present invention, the mesenchymal stem cell membrane biomimetic nanozyme has multiple therapeutic effects, including scavenging ROS, alleviating neuroinflammation caused by oxidative stress; regulating mitochondrial function and eliminating α-syn; inhibiting the M1 polarization of microglia induced by the inflammatory environment and promoting its transformation into the anti-inflammatory M2 phenotype, acting together to reduce brain nerve damage.

[0036] In summary, the mesenchymal stem cell membrane biomimetic nanozyme of the present invention has good biocompatibility, an extended in vivo circulation time, can actively target the Parkinson's disease lesion area, scavenge excessive accumulated ROS, relieve neuroinflammation caused by oxidative stress, and protect mitochondrial function by scavenging α-syn, regulate microglial polarization to play a role in protecting dopaminergic neurons, achieving the effect of treating Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification, and are used together with the following specific embodiments to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0038] Figure 1A is a transmission electron microscope image of the mesoporous polydopamine nanoparticles (mPDA) prepared in the preparation example;

[0039] Figure 1B is a transmission electron microscope image of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease prepared in the preparation example;

[0040] Figure 2 is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis image of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 1;

[0041] Figure 3 is a protein immunoblot image of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 2;

[0042] Figure 4 is an in vitro blood-brain barrier penetration image of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 3;

[0043] Figure 5 is an in vivo fluorescence distribution image (left) and its semi-quantitative image (right) of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in the treatment of Parkinson's disease in Detection Example 4;

[0044] Figure 6 is an open field experiment trajectory image (upper) and a quantitative image (lower) of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 5;

[0045] Figure 7 is a fluorescence image of the protective effect of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease on dopaminergic neurons in Detection Example 6;

[0046] Figure 8 is a fluorescence image of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease scavenging α-syn in Detection Example 7;

[0047] Figure 9 It is the result graph of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 8 to restore the DA level in the brain;

[0048] Figure 10 It is the result graph of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 9 to relieve oxidative stress in the brain, ROS (left), ATP (middle), MDA (right). Specific Embodiments

[0049] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0050] The following provides a detailed description of the solution of the present invention through specific preparation examples.

[0051] Among them, the raw materials used in the present invention are all conventional commercially available products, and will not be elaborated here.

[0052] The rats used in the present invention are provided by the animal experiment base of Nanjing Medical University; the C57 / B6 mice used for constructing the animal model are provided by the animal experiment base of Nanjing Medical University.

[0053] The bEnd.3 cells are purchased as a commercially available product with the catalog number TCM40 from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0054] Preparation Example Preparation of Mesenchymal Stem Cell Membrane Biomimetic Nanozyme for Targeted Treatment of Parkinson's Disease

[0055] The rats were sacrificed by decapitation and immersed in 75% ethanol. After 10 minutes, the limbs were dissected in a ventilated ultra-clean workbench. After removing the muscle tissue with scissors, the femurs, tibias, etc. were separated. The two ends of the bones were cut open with scissors and vertically placed into an EP tube with an open bottom for centrifugation to collect the mixed solution. The solution was filtered through a 70 μm cell filter membrane, and the cell suspension was collected and inoculated in MEM medium containing 10% fetal bovine serum and 100 IU / mL penicillin-streptomycin, and cultured in a constant temperature incubator at 37 °C and 5% CO2. After 48 hours, the medium was changed to remove other non-adherent cells, and the adherent MSC cells were continuously cultured. After about 5 passages, all the cells were collected for extracting cell membranes. The cell membranes were extracted by the method of repeated freezing and thawing: the solution containing cells was first placed in a -80 °C refrigerator for 5 minutes, then placed in a 37 °C shaker for 5 minutes, and this operation was repeated 3 times. Then, the supernatant was extracted by centrifugation at 3000 g for 5 minutes, and the supernatant was centrifuged at 50000 g for 30 minutes to obtain the precipitate, which was the mesenchymal stem cell membrane (denoted as MSCm), and it was stored in a -80 °C refrigerator for later use.

[0056] Dissolve 1.0 g of F-127 and 0.5 g of DA (i.e., dopamine hydrochloride, a conventional commercially available product with a purity of ≥98%) in 100 mL of a water and ethanol mixture with a volume ratio of 1:1, and stir vigorously at room temperature for 10 minutes to obtain a clear solution. Slowly inject 1.0 mL of TMB (tetramethylbenzene) into the solution, and stir at a constant speed of 500 rpm for 30 minutes to form a F-127 / TMB milky white nanoemulsion system. Then, gradually add 5.0 mL of concentrated ammonia water (NH4OH) dropwise to the above mixture to induce the self-polymerization of dopamine oligomers. After the addition is complete, continue the reaction for 2 hours. Centrifuge the formed dendritic mesoporous TMB / F127 / PDA polymer nanospheres at 16500 r for 15 minutes for separation, and wash them 3 times with an ultrasonic probe using water and ethanol in sequence to remove the soft template (i.e., the dendritic complex of F127 and TMB formed by the added excess F-127 and tetramethylbenzene). Finally, filter through a 0.45 μm microporous membrane and freeze-dry to obtain mesoporous polydopamine nanoparticles (denoted as mPDA) for standby.

[0057] Use a liposome extruder to extrude the collected MSC cell membranes, and successively select polycarbonate membranes with pore sizes of 400 nm and 200 nm to prepare MSC cell membranes with a particle size of approximately 200 nm. Mix the synthesized mesoporous polydopamine nanoparticles with mesenchymal stem cell membranes at a mass ratio of 50:1, and ultrasonicate in an ice bath for 5 minutes to coat the mesoporous polydopamine nanoparticles with MSC cell membranes. Centrifuge at 16500 r / min for 15 minutes to remove the unloaded MSC cell membranes, and obtain mesenchymal stem cell membrane biomimetic nanozymes (denoted as mPDA@MSC).

[0058] Characterize the morphology of the obtained mesenchymal stem cell membrane biomimetic nanozymes using a transmission electron microscope. The results are shown in Figure 1. It has a uniform spherical structure, and the outer layer is coated with mesenchymal stem cell membranes, with a particle size of approximately 200 nm.

[0059] Detection Example 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiment of mesenchymal stem cell membrane biomimetic nanozymes for targeted treatment of Parkinson's disease

[0060] Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the expression of total proteins and specific membrane proteins in mesenchymal stem cell membranes.

[0061] Specifically, the protein concentrations of the MSCm and mPDA@MSC prepared in the preparation example were measured using a BCA kit, and the protein concentrations in the MSCm and mPDA@MSC were kept consistent by adjusting the volumes of the MSCm and mPDA@MSC, respectively (i.e., the protein concentration in the adjusted MSCm solution was the same as that in the mPDA@MSC solution). 5xloading (conventional commercially available 5xSDS-PAGE gel loading buffer) was diluted to 1xloading with RIPA lysis buffer, and the marker and the MSCm solution and mPDA@MSC solution adjusted to the same protein concentration were diluted with it to an appropriate concentration (it should be noted that, because it will not be diluted to a certain concentration point value in actual operation, it is only necessary to ensure that the protein concentrations in the diluted MSCm solution and mPDA@MSC solution are each about 1 mg / mL, so as to ensure that the amount of protein after subsequent loading is not less than 20 μg), and the diluted MSCm solution and mPDA@MSC solution were placed in a 100°C air bath heater and heated for 10 minutes to denature the protein, and then cooled and centrifuged lightly to reduce the loss of the diluted MSCm solution and mPDA@MSC solution. Use a micropipette to draw 20 μL (the sample volume here is 20 μL, combined with the above-mentioned to ensure that the amount of protein after loading is not less than 20 μg, therefore, the protein concentration in the above-mentioned MSCm solution and mPDA@MSC solution is 1 mg / mL or slightly higher than 1 mg / mL), and slowly add the above-mentioned MSCm solution and mPDA@MSC solution to the sample well, and perform electrophoresis at a constant voltage of 160V for 60 minutes for protein separation. After the electrophoresis, stain with Coomassie Brilliant Blue dye, wash with distilled water 3 times after 15 minutes, and observe the distribution of protein bands under white light. It is consistent with the pure cell membrane, indicating that the mesenchymal stem cell membrane has been successfully coated. Results are shown in Figure 2 .

[0062] Test Example 2: Western blotting experiment of mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease

[0063] The same protein separation operation was performed according to the method of Detection Example 1. Then, the protein was transferred using a constant current of 300mA for 120min. After the protein was successfully transferred to the PVDF membrane, it was blocked with TBST buffer containing 5% skimmed milk powder and shaken at room temperature for 1h. After the membrane was washed with TBST 3 times for 5min each time, and then anti-CD47, VLA-4 and CXCR4 antibodies were added respectively, and incubated overnight at 4°C. After the incubation, the membrane was washed with TBST 3 times for 5min each time, and incubated with HRP-labeled secondary antibodies, and shaken at room temperature for 2h. After the membrane was washed with TBST, it was finally exposed in the Tanon 4600 series fully automatic chemiluminescence image analysis system. The protein immunoblot image is shown as follows Figure 3 As shown, Figure 3It was shown that the mesenchymal stem cell membrane-specific proteins CD47, CXCR4, and VLA-4 were successfully retained on the surface of the biomimetic nanozyme.

[0064] Detection Example 3: In vitro blood-brain barrier penetration experiment of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease

[0065] The ability of the biomimetic nanozyme to penetrate the blood-brain barrier was verified by Transwell experiment. bEnd.3 cells can express high levels of tight junction proteins and are thus particularly suitable for in vitro modeling of the blood-brain barrier (BBB). The bEnd.3 cells were seeded into the upper chamber of the Transwell cell culture at a density of 5×10 3 cells / well, placed in a 24-well plate, and 1 mL of DMEM medium was added to the lower chamber. After culturing routinely for 10 days, a monolayer of bEnd.3 cells with tight junctions was formed. Then, 40 μg / mL of rhodamine-fluorescently labeled mPDA and mPDA@MSC prepared in the Preparation Example were added to the upper chamber, and the culture was continued for a period of time. At different time points, the medium solution in the lower chamber was aspirated, and the fluorescence value at 576 nm under the excitation light of 545 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader, and normalization analysis was performed based on the fluorescence value of the pure DMEM medium at the initial time. The results obtained are as Figure 4 shown. At the same time, the fluorescence intensity in the lower chamber of the mPDA@MSC group was significantly stronger than that of the mPDA group, indicating that mPDA@MSC can penetrate the blood-brain barrier more effectively and enter the brain.

[0066] Detection Example 4: In vivo distribution and targeting investigation of the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease

[0067] MPTP was used to establish a Parkinson's animal model. Specifically, male 8-week-old C57 / B6 mice were subcutaneously injected with MPTP into the posterior neck for 5 consecutive days and then divided into two groups. The two groups of mice were respectively injected with Cy7-fluorescently labeled mPDA and mPDA@MSC prepared in the Preparation Example via the tail vein (i.e., one group of mice was injected with mPDA, denoted as the mPDA group; the other group of mice was injected with mPDA@MSC, denoted as the mPDA@MSC group). At 2 h and 8 h after administration, in vivo imaging was performed with a small animal in vivo imager to evaluate the brain targeting of the drug. After the imaging at the 8 h time point was completed, the mice were immediately sacrificed, and the main organs in the body (brain, heart, liver, spleen, lung, and kidney) were removed and further observed and imaged on the imager to further evaluate the targeting and the distribution of the drug in the organs of the mice, and quantitative analysis was performed using Living Image software through Region-Of-Interest (ROI) analysis. The results obtained are as Figure 5As shown. At different time points, the fluorescence intensity of the mPDA@MSC group was significantly increased compared with that of the mPDA group. Although the fluorescence decreased due to partial metabolism over time, the mPDA@MSC group still had strong fluorescence. This was mainly attributed to the coating of the mesenchymal cell membrane, which endows it with the ability to actively target the lesion site and have a long circulation in vivo. Moreover, the fluorescence images and semi-quantitative results of the isolated organs of mice after 8 h showed that the fluorescence in the brain of the mPDA@MSC group was significantly higher than that of the mPDA group, while less accumulation was observed in other organs such as the liver and kidney.

[0068] Open field test of mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease in Detection Example 5

[0069] An animal model was constructed according to the method of Detection Example 4, and drugs were administered before, after, and during the modeling process. Specifically, during the 5 days of constructing the animal model (i.e., the 5 days of injecting MPTP), drugs were administered every other day (3 times of administration), and drugs were administered once before and after the modeling, for a total of 5 times of drug administration to complete the modeling drug administration. At the same time, the animals and animal models were divided into 5 groups according to different drug-free, drug-administered, and drug types, namely the Control group (i.e., both MPTP during the modeling process and drugs during the drug administration process were replaced with normal saline, that is, the combination of normal saline + normal saline was used during the construction process), the MPTP group (i.e., the drug during the drug administration process was replaced with normal saline to construct the obtained animal model, that is, the combination of MPTP + normal saline was used during the construction process), the PDA group (the animal model constructed by administering PDA, the preparation of PDA was operated according to the method of preparing mPDA in the Preparation Example, the difference was that the template agent was not added, and polydopamine nanoparticles were synthesized, that is, the combination of MPTP + PDA was used during the construction process), the mPDA group (the animal model constructed by administering the PDA in the Preparation Example, that is, the combination of MPTP + mPDA was used during the construction process), and the mPDA@MSC group (the animal model constructed by administering the mPDA@MSC in the Preparation Example, that is, the combination of MPTP + mPDA@MSC was used during the construction process). After the modeling drug administration was completed, the motor function of the mice was evaluated through a series of behavioral experiments, such as the open field experiment, pole test, and rotarod test, and the mice should be trained 3 times before all the behavioral experiments officially started. Open field experiment: Prepare an open field with a length of 50 cm, a width of 50 cm, and a height of 40 cm in a quiet room. Before and after each experimental animal was used, the open field needed to be cleaned to remove irrelevant objects and residual odors. Before the experiment, the mice should be acclimated to the laboratory environment for at least 30 minutes to reduce the stress response to the new environment. At the start of the experiment, the mice were gently placed in the center or a corner of the open field device. After 2 minutes, a camera and motion tracking software were used to record and analyze the behavior of the animals, and the observation time was 5 minutes. After the experiment, analysis software was used to analyze the movement trajectory, total movement distance, and average movement speed of the mice within 5 minutes. The results are as Figure 6 shown. Representative movement trajectory diagrams showed that the movement of MPTP-treated mice was single, and also showed the shortest total movement distance within the area. Further analysis showed that the MPTP group had the lowest average movement speed. The results showed that the therapeutic effects of PDA and mPDA were weak, while the treatment with mPDA@MSC significantly improved the total movement distance and average speed, and increased the movement of the mice in the central area.

[0070] Detection Example 6 Protection experiment of mesenchymal stem cell membrane biomimetic nanozyme targeting the treatment of Parkinson's disease on dopaminergic neurons

[0071] The source of the brain slices is as follows: After the animal models constructed in Detection Example 5 or the animal models after drug administration are sacrificed after the aforementioned experiments are completed, the brains are taken and frozen sections are obtained.

[0072] The TH neurons in the brain were detected by immunofluorescence. The specific experimental operations are as follows: The brain slices were flatly attached to the glass slides, fixed with 4% paraformaldehyde for 30 minutes, gently washed 3 times with PBS, and then covered with a blocking solution containing freshly prepared 5% BSA and 10% goat serum, blocked at room temperature for 1 h, the blocking solution was removed, gently washed 3 times with PBS, and then the anti-TH antibody diluted with the blocking solution (1:200) was added and incubated overnight at 4°C. The next day, the primary antibody was discarded, gently washed 3 times with PBS, and the fluorescent secondary antibody diluted with the blocking solution was continuously added and incubated in the dark at room temperature for 2 h, the secondary antibody was discarded, and gently washed 3 times with PBS. Finally, the slides were sealed with an anti-fluorescence quencher containing DAPI, and the brain slices were placed under a fluorescence microscope for observation and photography. The results obtained are as Figure 7 shown. MPTP caused a significant reduction in TH neurons in the substantia nigra, and the treatment with mPDA@MSC could effectively reverse the depletion of dopaminergic neurons in the brain, showing more superior efficacy compared with the other two nanoparticles. The results indicate that mPDA@MSC can inhibit the decrease of TH, thereby further maintaining the DA level, and thus inhibiting the development of PD.

[0073] Detection Example 7 Experiment on the clearance of α-syn by mesenchymal stem cell membrane biomimetic nanozyme targeting the treatment of Parkinson's disease

[0074] The aggregation of α-syn was investigated by immunofluorescence. The specific experimental operations were as follows: The brain slices (the same source as the brain slices in Detection Example 6) were flatly attached to the glass slides, fixed with 4% paraformaldehyde for 30 minutes, gently washed 3 times with PBS, and then covered with a blocking solution containing freshly prepared 5% BSA and 10% goat serum, blocked at room temperature for 1 h, the blocking solution was removed, gently washed 3 times with PBS, and then the anti-α-syn antibody diluted with the blocking solution (1:200) was added and incubated overnight at 4°C. The next day, the primary antibody was discarded, gently washed 3 times with PBS, and the fluorescent secondary antibody diluted with the blocking solution was continuously added and incubated in the dark at room temperature for 2 h, the secondary antibody was discarded, and gently washed 3 times with PBS. Finally, the slides were sealed with an anti-fluorescence quencher containing DAPI, and the brain slices were placed under a fluorescence microscope for observation and photography. The results obtained are as Figure 8 shown. Compared with healthy mice, the level of α-syn in MPTP-induced PD mice was significantly increased, while in the drug treatment groups, especially the mPDA@MSC group, a significant decrease in the level of α-syn was observed, indicating that mPDA@MSC has significant advantages in clearing abnormally aggregated α-syn in the brain.

[0075] Detection Example 8: Detection of the Restoration of Brain DA Levels by Mesenchymal Stem Cell Membrane Biomimetic Nanozymes Targeting Parkinson's Disease

[0076] The mice in Detection Example 5 after MPTP modeling and drug treatment were sacrificed, and their brains were removed by decapitation. After rinsing the brain with PBS to remove excess blood on the brain surface, the brain was placed on ice. The striatum and midbrain were dissected and weighed, and a PBS solution containing protease inhibitor was added at a ratio of 10 μL per 1 mg. The brain tissue was gently minced into 1 cm3 pieces with scissors and then homogenized with a homogenizer for 5 minutes. After that, it was centrifuged at 15,000 rpm for 10 minutes at 4°C, and the supernatant was taken out. The dopamine content in the striatum was detected using a mouse dopamine ELISA kit. The results obtained are as Figure 9 shown. Compared with the MPTP group, PDA and mPDA can increase the DA level to a certain extent, while the DA level in the mice treated with mPDA@MSC increased significantly, almost similar to that of healthy mice. This finding is consistent with the results of TH immunofluorescence detection, indicating that mPDA@MSC can effectively protect dopaminergic neurons and increase the DA level in the brain.

[0077] Detection Example 9: Detection of the Alleviation of Intracerebral Oxidative Stress Indexes by Mesenchymal Stem Cell Membrane Biomimetic Nanozymes Targeting Parkinson's Disease

[0078] The supernatant after homogenizing the midbrain (from the same source as the brain homogenate in Detection Example 8) was used to detect the related indexes of the oxidative stress level in the midbrain of each group of mice using a mouse reactive oxygen species ELISA kit, a mouse adenosine triphosphate ELISA kit, and a mouse malondialdehyde ELISA kit. The results obtained are as Figure 10 shown. Compared with the control group, the ATP level in the MPTP group decreased significantly to about 52% of the healthy mouse level. mPDA@MSC could inhibit this decrease and restore it to 95% of the healthy mouse level. The restoration of the ATP level was consistent with the results of in vitro mitochondrial function experiments, indicating that mPDA@MSC has a good effect on protecting mitochondrial function both in vivo and in vitro. In addition, a significant increase in the ROS level was observed in MPTP-induced PD mice, reaching more than twice the level of healthy mice. At the same time, a similar abnormal increase in ROS was also found in the MDA detection results. Obviously, mPDA@MSC could significantly reduce the abnormally increased ROS and MDA, reducing them to 119% and 106% of the healthy group level, respectively. In summary, mPDA@MSC played an extremely important regulatory role in MPTP-induced mitochondrial dysfunction and oxidative stress.

[0079] In this invention, in vitro blood-brain barrier penetration experiments, in vitro ROS scavenging experiments, in vitro cell protection experiments, in vivo targeting studies, behavioral experiments, and in vivo pharmacodynamic studies were conducted on the mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease. It can be seen from the above detection examples that the mesenchymal stem cell membrane biomimetic nanozyme prepared in this invention can effectively target and deliver it to the lesion area, scavenge ROS, reduce brain damage caused by neuroinflammation, protect neurons, and play a role in treating Parkinson's disease.

[0080] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0081] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not further describe various possible combination methods.

[0082] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A mesenchymal stem cell membrane biomimetic nanozyme for targeted treatment of Parkinson's disease, characterized in that: The mesenchymal stem cell membrane bionic nanozyme comprises an inner core composed of mesoporous polydopamine nanoparticles and a mesenchymal stem cell membrane covering the inner core.

2. The mesenchymal stem cell membrane biomimetic nanozyme according to claim 1, characterized in that: The particle size of the mesenchymal stem cell membrane bionic nanozyme is 140-260nm.

3. A method for preparing the mesenchymal stem cell membrane biomimetic nanozyme according to claim 1, characterized in that: The preparation method comprises: S100, preparation of mesoporous polydopamine nanoparticles: dopamine hydrochloride and a surfactant are mixed in the presence of an ethanol aqueous solution, and then a template is added thereto to obtain a nanoemulsion; ammonia water is added to the obtained nanoemulsion to carry out a polymerization reaction to obtain a dendritic mesoporous polymer, and the precipitate is washed after centrifugation to remove excess surfactant and template, and the mixture is freeze-dried after microfiltration to obtain mesoporous polydopamine nanoparticles; S200, preparation of mesenchymal stem cell membrane bionic nanozyme: in an ice bath, the mesoporous polydopamine nanoparticles obtained in step S100 and the mesenchymal stem cell membrane are mixed in a weight ratio of 40-60:1, ultrasonically oscillated, and then centrifuged to remove the unencapsulated mesenchymal stem cell membrane to obtain the mesenchymal stem cell membrane bionic nanozyme.

4. The preparation method according to claim 3, characterized in that: In step S100, the surfactant is F-127 surfactant; And / or, the template agent is tetramethylbenzene; And / or, the weight ratio of the dopamine hydrochloride to the surfactant is 1:1.5-3, the volume ratio of ethanol to water in the ethanol aqueous solution is 1:0.8-1.2, and the amount of the ethanol aqueous solution is 80-120 mL relative to 1 g of the surfactant; And / or, relative to 1 g of the surfactant, the amount of the template agent used is 0.8-1.2 mL.

5. The preparation method according to claim 3 or 4, characterized in that: In step S100, the centrifugal speed of the centrifugal process is 15000-18000 r / min, and the centrifugal time is 12-18 min; and / or, the washing process is to use water and ethanol to perform ultrasonic washing alternately for 3 times; And / or, the filtration pore size of the microfiltration is 0.4-0.5 μm.

6. The preparation method according to claim 3 or 4, characterized in that: In step S200, the mesenchymal stem cell membrane is a mesenchymal stem cell membrane with a particle size of 140-260 nm obtained by extrusion using a liposome extruder.

7. The preparation method according to claim 3 or 4, characterized in that: The mesenchymal stem cell membrane is purified and amplified by a whole bone marrow adherence method and is extracted by a repeated freezing and thawing method.

8. The preparation method according to claim 7, characterized in that: The preparation method of the mesenchymal stem cell membrane specifically comprises: collecting a bone marrow suspension of a rat, inoculating it in a MEM culture medium containing fetal bovine serum and penicillin-streptomycin, culturing it in a constant temperature incubator, changing the medium after culturing for 42-54 hours to remove other non-adherent cells, continuing to culture the MSC cells that adhere to the wall, and collecting the cell-containing solution after culturing for 4-6 generations; placing the collected cell-containing solution in a -80°C refrigerator for 5 minutes, and then placing it in a 37°C shaker for 4-7 minutes, after continuous operation for 3-5 times, extracting the supernatant by centrifugation at 2500g-3500g for 3-8 minutes, and then centrifuging the supernatant at 40000g-60000g for 25-40 minutes to remove the precipitate, so as to obtain the mesenchymal stem cell membrane.

9. Use of the mesenchymal stem cell biomimetic nanozyme according to claim 1 in the preparation of a drug for treating Parkinson's disease.

10. A drug for targeted treatment of Parkinson's disease, characterized in that: The drug contains the mesenchymal stem cell membrane bionic nanozyme according to claim 1 or 2, or contains the mesenchymal stem cell membrane bionic nanozyme prepared by the preparation method according to any one of claims 3-8.

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