NK NANO pharmaceutical formulation, and preparation method therefor and use thereof

Abstract

The present application relates to the technical field of medicines. Disclosed are an NK nano pharmaceutical formulation, and a preparation method therefor and a use thereof. The NK nano pharmaceutical formulation comprises NK cells and a compound grafted on the surfaces of the NK cells and having a structural formula of (I).

Description

NK nanopharmaceutical preparation and its preparation method and application Technical Field

[0001] The present application belongs to the field of pharmaceutical technology, and particularly relates to NK nanopharmaceutical preparations, preparation methods, and applications thereof. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder in middle-aged and elderly people. Its main clinical symptoms include resting tremor, bradykinesia, increased muscle tone, and postural balance disorders. With the aging of the population, the incidence of PD is increasing annually. Epidemiological data show that the prevalence of the disease in those aged 65 and above is 1.67%. The main pathological changes of PD are the progressive degeneration and death of dopaminergic (DA) neurons in the substantia nigra of the midbrain, and the appearance of eosinophilic protein inclusions formed by aggregates of α-synuclein within the remaining neurons. The etiology of PD is currently unknown, but most scholars believe that PD is a multifactorial disease caused by both genetic and environmental factors. To date, abnormal expression of multiple genes has been found to be associated with PD, among which α-synuclein encoded by PARK1 / SNCA is one of the proteins most closely related to PD (References: (1) Shahmoradian SH, Lewis AJ, Genoud C, Hench J, Moors TE, Navarro PP, et al. Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes. Nature neuroscience. 2019; 22: 1099-109. (2) Kalia LV, Lang AE. Parkinson's disease. Lancet. 2015; 386: 896-912. (3) Shahnawaz M, Mukherjee A, Pritzkow S, Mendez N, Rabadia P, Liu X, et al. Discriminating alpha-synuclein strains in Parkinson's disease and multiple system atrophy. Nature. 2020; 578: 273-7. (4) Fusco G, Chen SW, Williamson PTF, Cascella R, Perni M, Jarvis JA, et al. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers. Science. 2017; 358: 1440-3). Autophagy dysfunction and oxidative stress damage are important mechanisms in the pathogenesis of PD.In recent years, a variety of small molecule drugs that promote autophagy and resist oxidative stress, such as inhibitors of NLRP3 and RIPK, have been shown to protect DA neuron damage in PD (see references: (5) Xu D, Zhao H, Jin M, Zhu H, Shan B, Geng J, et al. Modulating TRADD to restore cellular homeostasis and inhibit apoptosis. Nature. 2020; 587: 133-8. (6) Han X, Sun S, Sun Y, Song Q, Zhu J, Song N, et al. Small molecule-driven NLRP3 inflammation inhibition via interplay between ubiquitination and autophagy: implications for Parkinson disease. Autophagy. 2019; 15: 1860-81. (7) Cheng J, Liao Y, Dong Y, Hu H, Yang N, Kong X, et al. Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice. Autophagy. 2020;16:2193-205.). However, the extent to which these drugs improve α-synuclein pathology remains unclear, and the safety and efficacy of these drugs in treating PD remain under investigation.Many natural drugs, such as mangiferin, squalamine, and tea polyphenols, have also been reported to reduce oxidative stress damage, mitochondrial dysfunction, and immune inflammatory response caused by α-synuclein, thereby protecting dopamine neurons and exerting neuroprotective effects in PD (see references: (8) Feng ST, Wang ZZ, Yuan YH, Sun HM, Chen NH, Zhang Y. Mangiferin: A multipotent natural product preventing neurodegeneration in Alzheimer's and Parkinson's disease models. Pharmacological research. 2019; 146: 104336. (9) Perni M, Galvagnion C, Maltsev A, Meisl G, Muller MB, Challa PK, et al. A natural product inhibits the initiation of alpha-synuclein aggregation and suppresses its toxicity. Proc Natl Acad Sci US A. 2017; 114: E1009-E17. (10) Zhou ZD, Xie SP, Saw WT, Ho PGH, Wang H, Lei Z, et al. The Therapeutic Implications of Tea Polyphenols Against Dopamine(DA) Neuron Degeneration in Parkinson's Disease(PD). Cells. 2019; 8.).In 2019, Madeo Frank's team first reported that a natural flavonoid drug derived from tomorrow leaf - 4,4'-dimethoxychalcone (DMC) can activate GATA transcription factors independently of the mTORC1 signaling pathway, promote autophagy levels in different species such as yeast, nematodes, fruit flies, mice and primates, and thus exert a protective effect (see reference: (11) Carmona-Gutierrez D, Zimmermann A, Kainz K, Pietrocola F, Chen G, Maglioni S, et al. The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species. Nature communications. 2019; 10: 651.). Tomorrow leaf is produced in Japan, and its efficacy and main indications are recorded in detail in the Ming Dynasty Li Shizhen's "Compendium of Materia Medica" and the Japanese "Yamato Honma". Modern pharmacological research has revealed that ashitaba contains multiple active ingredients, including flavonoids, choline, and coumarins, the most prominent of which are chalcone compounds. Chalcone, a type of flavonoid, possesses significant antioxidant properties. DMC significantly promotes autophagy and has a relatively good safety profile. However, its bioavailability is low, its targeting is weak, and its effect on α-synuclein pathology in Parkinson's disease is not significant.

[0003] Therefore, there is an urgent need to provide a new drug that can improve its efficiency in crossing the blood-brain barrier, increase its bioavailability, clear α-synuclein inside and outside cells, and improve behavioral disorders and brain pathological damage in Parkinson's disease models.

[0004] Summary of the Invention

[0005] The present application aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present application proposes a NK nano drug preparation and its preparation method and application. The preparation method of the NK nano drug preparation described in the present application is simple and rapid to operate, and the prepared NK nano drug preparation has stable properties and high safety, which improves the problems of low permeability of DMC (4,4'-dimethoxychalcone) through the blood-brain barrier, poor targeting, and no significant pathological effect on α-synuclein in PD. In the application of the NK nano drug preparation of the present application in the treatment of Parkinson's disease, after passing through the blood-brain barrier, under the environment of oxidative stress damage in the brain, the NK cells and targeted small molecule drugs in the NK nano drug preparation are released respectively, and the motor behavior disorders of Parkinson's mice are improved by clearing α-synuclein inside and outside the cells, and can be used as a new type of neuroprotective drug in the treatment of anti-neurodegenerative diseases.

[0006] According to the technical solution of the present application, the lead compound in the NK nanopharmaceutical preparation of the present application has significant autophagy effect, good α-synuclein clearance effect, and no significant toxicity (the compound is named 2-Propen-1-one,2-fluoro-1,3-bis(4-methoxyphenyl)-,(E)-,C 17 H 15 FO3, with a molecular weight of 286.30, is named F-DMC). Studies on the pharmacological activity of F-DMC have confirmed its significant anti-PD effect. Combined with cell therapy, natural killer cells (NK cells) and F-DMC are grafted to promote the smooth passage of F-DMC through the blood-brain barrier. Under conditions of oxidative stress damage in the brain, the connection between NK cells and F-DMC is severed, and the released NK cells can clear extracellular α-synuclein. F-DMC taken up by dopamine neurons can clear intracellular α-synuclein by promoting autophagy. Under this dual action, the clearance and degradation of pathological α-synuclein are completed, thereby improving the behavioral disorders and brain pathological damage of the PD model. This treatment model is expected to provide new reference for the treatment of neurodegenerative diseases.

[0007] The first aspect of the present application provides a NK nanodrug preparation.

[0008] Specifically, the NK nano drug preparation includes NK cells and C grafted on the surface of the NK cells. 17 H 15 FO3.

[0009] The C 17 H 15 The structural formula of FO3 is

[0010] Preferably, the NK nano drug preparation further comprises polylactic acid-glycolic acid copolymer and / or active oxygen responsive phospholipid. 17 H 15 FO3 is coated with poly(lactic-co-glycolic acid) copolymer and / or reactive oxygen species-responsive phospholipids and then grafted with NK cells.

[0011] Preferably, the weight average molecular weight of the polylactic acid-glycolic acid copolymer is 30k-60k, preferably 45k-55k, more preferably 48k-50k.

[0012] Preferably, in the polylactic acid-glycolic acid copolymer, the molar ratio of the polylactic acid structure to the glycolic acid structure is 1:(0.8-1.2), preferably 1:1.

[0013] Preferably, the reactive oxygen species responsive phospholipid (abbreviated as ROS responsive phospholipid) is distearoylphosphatidylethanolamine-polyethylene glycol.

[0014] Preferably, the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is at least one of polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 6000, preferably polyethylene glycol 2000. The stearoylphosphatidylethanolamine-polyethylene glycol is an active oxygen responsive phospholipid.

[0015] The second aspect of the present application provides a method for preparing a NK nanoparticle drug preparation.

[0016] Specifically, the preparation method of the NK nanopharmaceutical preparation comprises the following steps:

[0017] (1) Take C 17 H 15 FO3, poly(lactic-co-glycolic acid) and / or active oxygen responsive phospholipids were mixed with an organic solvent, ultrasonically dispersed, added with water, and dialyzed to prepare NPs@F-DMC;

[0018] (2) mixing the targeting peptide RVG29 with the NPs@F-DMC to prepare RVG-NPs@F-DMC;

[0019] (3) The NK cells were added to a phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride, incubated, and washed to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride. The RVG-NPs@F-DMC was then mixed with the NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride and incubated to prepare a NK nanoparticle drug preparation (denoted as NK cell-NPs@F-DMC).

[0020] Preferably, in step (1), the C 17 H 15 The mass ratio of FO3, poly(lactic acid-glycolic acid copolymer), and active oxygen responsive phospholipid is 1:(0.8-3):(0.8-3), preferably 1:(1-2):(1-2).

[0021] Preferably, in step (1), the organic solvent comprises ethanol and / or dimethyl sulfoxide. When the organic solvent is a mixture of ethanol and dimethyl sulfoxide, the volume ratio of ethanol to dimethyl sulfoxide is 1:(7-10), preferably 1:9.

[0022] Preferably, in step (1), the C 17 H 15 The mass volume ratio of FO3 to the organic solvent is 1 mg: (0.1-1) mL, preferably 1 mg: (0.1-0.5) mL.

[0023] Preferably, in step (1), the power of the ultrasound is 20-50W, preferably 30-35W.

[0024] Preferably, in step (1), the water is ultrapure water.

[0025] Preferably, in step (1), water is added dropwise during ultrasonic dispersion.

[0026] Preferably, in step (1), the volume of water is 1-20 mL, preferably 2-10 mL.

[0027] Preferably, in step (1), the dialysis is performed using a dialysis bag and ultrapure water for 40-48 hours.

[0028] More preferably, the molecular weight cut-off of the dialysis bag is 3000Da-3500Da. The function of dialysis is to remove C 17 H 15 FO3 and organic solvents.

[0029] Preferably, in step (2), 1-5 μg of the targeting peptide RVG29 is mixed with the NPs@F-DMC prepared in step (1) for 0.5-4 hours, and ultrafiltration is performed to remove the free targeting peptide RVG29 to obtain the RVG-NPs@F-DMC, which is then stored below 4°C for future use.

[0030] Preferably, in step (3), the concentration of tris(2-carboxyethyl)phosphine hydrochloride in the phosphate buffered saline solution containing tris(2-carboxyethyl)phosphine hydrochloride is 0.1-1 mg / mL, preferably 0.1-0.5 mg / mL.

[0031] Preferably, in step (3), the volume of the phosphate buffered saline solution containing tris(2-carboxyethyl)phosphine hydrochloride is 1-15 mL, preferably 1-10 mL.

[0032] Preferably, in step (3), the number is (1-10)×10 6 NK cells were added to phosphate-buffered saline containing tris(2-carboxyethyl)-phosphine hydrochloride.

[0033] Preferably, in step (3), the incubation temperature is 35-37° C., and the incubation time is 5-50 minutes, preferably the incubation temperature is 37° C., and the incubation time is 10-45 minutes. After incubation, the cells are washed 2-10 times with phosphate buffered saline.

[0034] Preferably, in step (3), 1 mL of RVG-NPs@F-DMC is mixed with NK cells treated with tris(2-carboxyethyl)phosphine hydrochloride. After mixing, the concentration of RVG-NPs@F-DMC is 10-50 μg / mL, preferably 5-60 μg / mL.

[0035] Preferably, in step (3), before preparing the NK nano drug preparation, the mixture is washed 2-10 times with phosphate buffered saline.

[0036] Preferably, in step (3), after incubation, the mixture is centrifuged 2-10 times and dispersed with phosphate buffered saline to remove impurities such as free RVG29 and tris(2-carboxyethyl)phosphine hydrochloride to prepare a NK nanopharmaceutical preparation.

[0037] Preferably, the preparation method of the NK nanopharmaceutical preparation comprises the following steps:

[0038] (1) Take C 17 H 15 FO3, poly(lactic acid-co-glycolic acid), active oxygen responsive phospholipids were mixed with organic solvents, ultrasonically dispersed, added with water, and dialyzed to prepare NPs@F-DMC; C 17 H 15 The mass ratio of FO3, poly(lactic-co-glycolic acid) copolymer, and active oxygen responsive phospholipid is 1:(0.8-3):(0.8-3), C 17 H 15 The mass volume ratio of FO3 to organic solvent is 1 mg: (0.1-1) mL. Dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500 Da and ultrapure water for 40-48 hours. The ultrasonic power is 20-50 W.

[0039] (2) 1-5 μg of the targeting peptide RVG29 was mixed with the NPs@F-DMC prepared in step (1) for 0.5-4 hours, and ultrafiltration was performed to obtain RVG-NPs@F-DMC;

[0040] (3) Set the quantity to (1-10)×10 6NK cells were added to a phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride, incubated at 35-37°C for 5-50 minutes, and washed 2-10 times with the phosphate buffered saline solution to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride; then 1 mL of RVG-NPs@F-DMC was mixed with the NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride, incubated at 35-37°C for 5-50 minutes, centrifuged, and washed 2-10 times with the phosphate buffered saline solution to prepare an NK nanoparticle preparation (denoted as NK cell-NPs@F-DMC); wherein the concentration of tris(2-carboxyethyl)-phosphine hydrochloride in the phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride was 0.1-1 mg / mL, and the volume of the phosphate buffered saline solution was 1-15 mL.

[0041] The third aspect of the present application provides the application of NK nano drug preparations.

[0042] Application of the above-mentioned NK nanopharmaceutical preparation in the preparation of drugs for treating neurodegenerative diseases.

[0043] Preferably, the neurodegenerative disease includes Parkinson's disease.

[0044] A drug for treating Parkinson's disease, comprising the above-mentioned NK nano drug preparation.

[0045] Compared with the prior art, the beneficial effects of this application are as follows:

[0046] In the NK nano drug preparation of the present application, natural killer cells (natural killer cell, NK cell) and F-DMC are grafted to promote the smooth passage of F-DMC through the blood-brain barrier. Under the conditions of oxidative stress damage in the brain, the connection between NK cells and F-DMC is cut off, and the released NK cells can remove extracellular α-synuclein, and the F-DMC taken up by dopamine neurons can remove intracellular α-synuclein by promoting autophagy. Under the dual effect, the removal and degradation of pathological α-synuclein are completed, thereby improving the behavioral disorders and brain pathological damage of the PD model. This treatment model is expected to provide a new reference for the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a structural diagram of F-DMC and DMC.

[0048] Figure 2 shows that F-DMC significantly promotes the autophagy level of dopaminergic neuron MN9D cells.

[0049] FIG3 shows the effect of F-DMC significantly promoting the clearance of pathological α-synuclein in dopaminergic neuron MN9D cells.

[0050] Figure 4 shows the particle size detection results of NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1.

[0051] FIG5 is a scanning electron micrograph of NK cells and the NK nanoparticle drug preparation prepared in Example 1.

[0052] Figure 6 shows the ROS-responsive drug release performance test results of the labeled NK nanomedicine preparation (NK cell-NPs@ICG).

[0053] FIG7 shows the results of the NK nanomedicine preparation of Example 1 treating and improving the motor behavior disorder in AAV-A53Tα-Syn model mice.

[0054] FIG8 shows the results of the NK nanomedicine preparation of Example 1 treating and improving dopamine neuron damage in AAV-A53Tα-Syn model mice.

[0055] FIG9 shows that the NK nanomedicine preparation of Example 1 reduces the pathological α-synuclein expression in AAV-A53Tα-Syn model mice. DETAILED DESCRIPTION

[0056] In order to make the technical solution of this application more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed in this application.

[0057] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0058] Example 1: Preparation of NK nanopharmaceutical preparations

[0059] The preparation method of NK nano drug preparation comprises the following steps:

[0060] (1) Take 5 mg C 17 H 15FO3 (F-DMC, purchased from ChemDiv, USA), 10 mg of poly(lactic acid-co-glycolic acid) (the weight-average molecular weight of poly(lactic acid-co-glycolic acid) is 50k, and the molar ratio of poly(lactic acid) structure to glycolic acid structure in poly(lactic acid-co-glycolic acid) is 1:1), 10 mg of distearoylphosphatidylethanolamine-polyethylene glycol (the polyethylene glycol in distearoylphosphatidylethanolamine-polyethylene glycol is polyethylene glycol 2000) and 1 mL of organic solvent (the organic solvent is a mixture of ethanol and dimethyl sulfoxide, and the volume ratio of ethanol to dimethyl sulfoxide is 1:9) were mixed, and 5 mL of ultrapure water was added dropwise under ultrasonic conditions with a power of 30 W to disperse the mixture, and then dialyzed (dialysis was carried out using a dialysis bag and dialyzed with ultrapure water for 48 hours, and the molecular weight cut-off of the dialysis bag was 3500 Da) to prepare NPs@F-DMC;

[0061] (2) 2 μg of the targeting peptide RVG29 was mixed with the NPs@F-DMC prepared in step (1) for 2 hours, and the free targeting peptide RVG29 was ultrafiltered to obtain RVG-NPs@F-DMC;

[0062] (3) Set the quantity to 5×10 6 NK cells were added to a phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride (the concentration of tris(2-carboxyethyl)-phosphine hydrochloride was 0.3 mg / mL, the volume of phosphate buffered saline solution was 5 mL, and phosphate buffered saline solution was referred to as PBS), incubated at 37°C for 20 minutes, and washed three times with phosphate buffered saline to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride. Then, 1 mL of RVG-NPs@F-DMC (concentration 20 μg / mL) was mixed with 1×10 5 NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride were mixed, incubated at 37°C for 30 minutes, and washed three times with phosphate buffered saline to prepare NK nanoparticle drug preparation (denoted as NK cell-NPs@F-DMC).

[0063] Example 2: Preparation of NK nanopharmaceutical preparations

[0064] The preparation method of NK nano drug preparation comprises the following steps:

[0065] (1) Take 5 mg C 17 H 15FO3(F-DMC), 5 mg of poly(lactic acid-co-glycolic acid) (the weight average molecular weight of poly(lactic acid-co-glycolic acid) is 50k, and the molar ratio of poly(lactic acid) structure to glycolic acid structure in poly(lactic acid-co-glycolic acid) is 1:1), 5 mg of distearoylphosphatidylethanolamine-polyethylene glycol (the polyethylene glycol in distearoylphosphatidylethanolamine-polyethylene glycol is polyethylene glycol 2000) and 1 mL of organic solvent (the organic solvent is a mixture of ethanol and dimethyl sulfoxide, and the volume ratio of ethanol to dimethyl sulfoxide is 1:9) were mixed, and 10 mL of ultrapure water was added dropwise under ultrasonic conditions with a power of 30 W to disperse the mixture, and then dialyzed (dialysis was carried out using a dialysis bag, and dialysis was carried out using ultrapure water for 48 hours, and the molecular weight cutoff of the dialysis bag was 3500 Da) to prepare NPs@F-DMC;

[0066] (2) 2 μg of the targeting peptide RVG29 was mixed with the NPs@F-DMC prepared in step (1) for 1 hour, and the free targeting peptide RVG29 was ultrafiltered to obtain RVG-NPs@F-DMC;

[0067] (3) Set the quantity to 2×10 6 NK cells were added to a phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride (the concentration of tris(2-carboxyethyl)-phosphine hydrochloride was 0.1 mg / mL, and the volume of the phosphate buffered saline solution was 3 mL), incubated at 37°C for 45 minutes, and washed three times with phosphate buffered saline to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride. Then, 1 mL of RVG-NPs@F-DMC (concentration of 20 μg / mL) was mixed with 1×10 5 NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride were mixed, incubated at 37°C for 30 minutes, and washed three times with phosphate buffered saline to prepare NK nanoparticle drug preparation (denoted as NK cell-NPs@F-DMC).

[0068] The product effect testing method is as follows:

[0069] 1. ROS-responsive drug release experiment of NK nanoparticles

[0070] A near-infrared fluorescent dye ICG with a mass ratio of 2% was used instead of F-DMC to prepare a labeled nanoparticle drug preparation (denoted as RVG-NPs@ICG), and a labeled NK nanoparticle drug preparation (denoted as NK cell-NPs@ICG) was further prepared. The labeled NK nanoparticle drug preparation (NK cell-NPs@ICG) was incubated with a culture medium containing ROS (400mM H2O2 and 3.2μM copper chloride) and a culture medium without ROS at 37°C. Low-speed centrifugation (300g, 3 minutes) was performed at different time points, and a certain volume of supernatant was taken to measure its fluorescence intensity. The same volume of culture medium was added to the co-incubation culture dish, and the NPs@ICG release at different time points was calculated by the following formula. The results are shown in Figure 6.

[0071] NPs@ICG cumulative release rate

[0072] Where: V e is the displacement volume, V o is the volume of the released liquid, F i is the fluorescence intensity in the release liquid during the i-th displacement sampling.

[0073] 2. Cell Viability Assay

[0074] (1) Add 100 μL of cell suspension to a 96-well plate, with 5000 cells per well.

[0075] (2) After culturing in a cell culture incubator for 24 hours, the cells were subjected to experimental treatment.

[0076] (3) After the time is up, discard the supernatant, prepare the working solution of CCK-8 detection reagent and complete culture medium at a ratio of 1:9, completely remove the old culture medium, add 100 μL of working solution to each well, and incubate in a cell culture incubator.

[0077] (4) After incubation for 1 h, the absorbance was measured at a wavelength of 450 nm.

[0078] 3. Electron microscopy

[0079] The experimental steps for transmission electron microscopy are as follows:

[0080] (1) Fix with 2.5% glutaraldehyde in 0.1 M PBS at room temperature for 1 hour, and rinse three times with 0.1 M PBS.

[0081] (2) Stain with 1% osmium tetroxide for 30 minutes and rinse with distilled water three times, 15 minutes each time.

[0082] (3) Dehydrate with 50%, 70%, 90% and 100% alcohol at 4°C, 15-20 minutes for each gradient.

[0083] (4) Acetone / embedding agent (1:1), room temperature for 1 hour, acetone / embedding agent (1:3), 4°C overnight, pure embedding agent, 4°C overnight.

[0084] (5) The resin-infiltrated samples were cured in an oven at 65 °C for 24 h.

[0085] (6) Ultramicrotome slices, thickness 50-100 nm.

[0086] (7) 3% uranyl acetate-lead citrate double staining.

[0087] (8) Transmission electron microscopy observation and photography.

[0088] 4. PD Model Preparation and Drug Treatment

[0089] The PD model utilizes brain-targeted injection of AAV-A53Tα-Syn virus. The steps for brain-targeted injection are as follows: After C57BL / 6J mice are anesthetized with isoflurane inhalation, they are fixed in a prone position on a stereotaxic instrument, with the incisors 2.3 mm below the ear bars and the anterior and posterior bregma aligned. Hair is trimmed, the skin is disinfected with iodine, and the scalp is incised and the periosteum is peeled. The coordinates of the substantia nigra are 3.0 mm posterior to the anterior bregma, 1.3 mm lateral to the midline, and 4.7 mm below the periosteum. 0.5 μL of control virus or AAV-A53Tα-Syn virus is injected. After injection, the needle is withdrawn for 10 minutes, and one stitch is sutured. The mice are kept warm. Penicillin is injected intramuscularly. After awakening, the mice are placed in a cage for feeding. One month after viral injection, the animals were treated with PBS, NK cells, nanoparticle-encapsulated F-DMC (NPs@F-DMC), and NK cell-NPs@F-DMC via the tail vein every other day for a total of eight treatments. Following the final treatment, behavioral and other assessments were performed.

[0090] 5. Behavioral Experiments

[0091] (1) Open field test: The open field box is a square open box with a length of 50 cm, a width of 50 cm, and a height of 40 cm, and the inner wall is painted black. The top of the open box is artificially illuminated. The open box is divided into a central area (length of 25 cm, width of 25 cm) and a peripheral area. At the beginning of the experiment, the mouse is placed in the center of the open box, and the position and direction of each mouse are kept consistent. The mouse is allowed to move freely in the open box for 15 minutes. The movement speed of the mouse is recorded using Smart 3.0 video tracking software.

[0092] (2) Roller test: The roller test requires animals to maintain balance and move continuously on the roller. It is a widely used test to detect movement coordination. The roller diameter is 6 cm and the rotation speed is 20 r / min. After the mice adapt to it 5 times, the interval between each test is 1 minute. The average value of 5 consecutive tests is taken.

[0093] (3) Pole climbing test: A 2cm diameter foam plastic ball was fixed to the top of a 50cm long and 1cm diameter wooden pole. Two layers of gauze were wrapped around the pole to prevent slipping. The mouse was held by its tail and placed head down on the top of the pole (with both hind legs of the mouse placed on the ball). The mouse was allowed to climb down naturally. The mouse was considered to have climbed the full length from standing on the top of the pole to both forelegs touching the platform at the bottom of the pole. After modeling, a certain number of mice were selected from each group to perform the pole climbing test. The behavioral changes of the mice were observed and the climbing time was recorded.

[0094] (4) Hanging test: A homemade organic glass test box was used. A horizontal metal rod with a diameter of 1.5 mm was placed 30 cm above the ground. A cover was placed 1 cm above the metal rod to prevent the mouse from sitting on the metal rod. During the experiment, the mouse was suspended on the metal rod with its front paws holding the metal rod for 10 seconds. The hanging score was calculated as follows: 3 points for a mouse holding the rod with both hind paws; 2 points for a mouse holding the rod with one hind paw; 1 point for a mouse failing to hold the rod with both hind paws; and 0 points for a mouse falling down.

[0095] 6. Western Blot Hybridization Experiment

[0096] Extract a protein sample from the cells and determine the protein concentration using a BCA kit. Add loading buffer in a specific ratio and incubate in a boiling water bath at 100°C for 5 minutes. After electrophoresis, transfer the protein to a PVDF (polyvinylidene fluoride) membrane. Rinse the PVDF membrane with TBST buffer (Tris-buffered saline) for 5 minutes at room temperature, block with 5% bovine serum albumin for 2 hours, and then rinse three times with TBST buffer for 5 minutes each. Incubate with the primary antibody overnight at 4°C. Rinse the membrane three times with TBST buffer for 5 minutes each, then incubate with the secondary antibody at room temperature for 1 hour and rinse with TBST buffer. Mix equal amounts of chemiluminescent reagents A and B, incubate the PVDF membrane for 1 minute, and expose it on an imager.

[0097] 7. Immunohistochemical Staining

[0098] Mice were anesthetized with isoflurane inhalation and perfused with pre-chilled saline and 4% paraformaldehyde. Mouse brains were fixed in 4% paraformaldehyde at 4°C for approximately 6 hours. The brains were then dehydrated in a gradient of 20% and then 30% sucrose solutions in a refrigerator at 4°C. The tissues were embedded in OCT embedding medium and serial coronal sections were cut using a constant-temperature cryostat. Sections were placed in a staining jar containing 0.01 M citrate buffer and incubated at 98°C for 10–15 minutes. The sections were then cooled to room temperature and rinsed three times with PBS for 5 minutes each. The membranes were blocked with 0.3% Triton and 5% BSA for 30 minutes and then rinsed three times with PBS for 5 minutes each. Primary antibody was added and the sections were incubated overnight at 4°C. The next day, sections were removed from the refrigerator and warmed to room temperature for 45 minutes. Secondary antibody was added to the sections and incubated at 37°C for 60 minutes. The sections were washed three times with PBS for 5 minutes each. DAB colorimetric solution was added to the sections and the reaction time was determined under a microscope at room temperature. The sections were then rinsed with running tap water. The sections were counterstained with hematoxylin for 2 min, separated with hydrochloric acid and alcohol once, rinsed with running water, dehydrated with graded alcohol, mounted with gum, and examined under a microscope.

[0099] 8. Immunofluorescence Staining

[0100] After rinsing with PBS, sections were permeabilized and blocked with 0.3% Triton and 5% BSA in PBS at 37°C for 1 hour. Primary antibodies were then incubated overnight at 4°C. After three washes with PBS, sections were incubated with secondary antibodies for 1 hour, dried in the shade, and mounted with glycerol. Scanning was performed under a laser confocal microscope, and digital imaging was performed using a computer for data acquisition.

[0101] The product effect test results are as follows:

[0102] 1. Cellular Experimental Verification of F-DMC-Promoted Autophagy

[0103] MN9D dopamine neuron cell lines were cultured and treated with various concentrations of F-DMC (0, 5, 10, 50, and 100 μM), with rapamycin (10 μM) used as a positive control. After 24 hours of culture, the cells were observed by immunoblotting and electron microscopy. The results are shown in Figure 2 ("Ctrl" in Figure 2 indicates the rapamycin positive control; LC3Ⅰ, LC3Ⅱ, p62, and GAPDH indicate proteins).

[0104] Figure 1 is a structural diagram of F-DMC and DMC; Figure A in Figure 1 represents the two-dimensional structure of DMC, and Figure B represents the two-dimensional structure of F-DMC.

[0105] Figure 2 shows that F-DMC significantly promotes the level of autophagy in dopaminergic neuron MN9D cells. As can be seen from Figure 2, F-DMC can promote the production of autophagy in MN9D cells in a concentration-dependent manner, specifically as an increase in the LC3Ⅱ / Ⅰ ratio (see Figure 2, Panel A). In addition, at the same concentration (100μM), F-DMC significantly promotes autophagy production compared to DMC (see Figure 2, Panel B). Transmission electron microscopy observation showed that F-DMC significantly promoted the formation of autophagic lysosomes, specifically as an increase in the number of lysosomes (see Figure 2, Panel C). Cell viability experiments showed that F-DMC at different concentrations did not show cytotoxicity, confirming the safety of the drug (see Figure 2, Panel D).

[0106] 2. F-DMC significantly promotes the clearance and degradation of pathological α-synuclein

[0107] MN9D cells were treated with human α-synuclein fibrils (hα-Syn), and F-DMC and DMC (100 μM) were added and cultured for 24 hours. The results are shown in FIG3 .

[0108] Figure 3 ("Ctrl" in Figure 3 indicates a rapamycin positive control) shows the effect of F-DMC on significantly promoting the clearance of pathological α-synuclein in dopaminergic neuron MN9D cells. As can be seen from Figure 3, compared with DMC, F-DMC can significantly clear α-synuclein fibrils (manifested by reduced expression of phosphorylated α-synuclein) (see Figure 3, Panel A). In addition, 50μM and 100μM F-DMC can significantly clear phosphorylated α-synuclein and reduce endogenous α-synuclein expression, demonstrating a strong α-synuclein degradation effect (see Figure 3, Panel B).

[0109] 3. Characterization of NK Nanomedicine Intermediates

[0110] The particle sizes of NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1 were measured using a Zetasizer Nano ZS instrument, and the results are shown in FIG4 .

[0111] Figure 4 shows the particle size measurement results of NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1. As shown in Figure 4 (where "Size" indicates size and "PDI" indicates polydispersity index), the particle size distribution of NPs@F-DMC and RVG-NPs@F-DMC ranges from 80 to 300 nm (see Figure 4, Panel A), with average particle sizes of 160.0 ± 6.9 nm and 167.3 ± 6.5 nm (see Figure 4, Panel B). The PDI polydispersity indexes are 0.445 and 0.403, respectively (see Figure 4, Panel C).

[0112] 4. Scanning electron microscopy characterization results of NK nanoparticles

[0113] Figure 5 is a scanning electron micrograph of NK cells and the NK nanoparticle drug preparation prepared in Example 1; "NK cell" in Figure 5 represents NK cells, and "NK cell-NPs@F-DMC" represents the NK nanoparticle drug preparation prepared in Example 1. The drugs grafted and encapsulated on the surface of NK cells are shown by arrows in Figure 5.

[0114] 5. ROS-responsive drug release experiment of NK nanoparticles

[0115] A near-infrared fluorescent dye ICG with a mass ratio of 2% was used instead of F-DMC. Referring to the method of Example 1, a fluorescently labeled nano drug preparation (denoted as RVG-NPs@ICG) was prepared, and a fluorescently labeled NK nano drug preparation (denoted as NK cell-NPs@ICG) was further prepared. The fluorescently labeled NK nano drug preparation (NK cell-NPs@ICG) was incubated with a culture medium containing ROS (400mM H2O2 and 3.2μM copper chloride) and a culture medium without ROS at 37°C. Low-speed centrifugation (300g, 3 minutes) was performed at different time points, a certain volume of supernatant was taken to measure its fluorescence intensity, and the same volume of culture medium was added to the co-incubation culture dish. The cumulative release rate of NPs@ICG at different time points was calculated by the following formula, and the results are shown in Figure 6.

[0116] NPs@ICG cumulative release rate

[0117] Where: V e is the displacement volume, V o is the volume of the released liquid, F i is the fluorescence intensity in the release liquid during the i-th displacement sampling.

[0118] Figure 6 shows the ROS-responsive drug release performance test results of the fluorescently labeled NK nanoparticle drug preparation (NK cell-NPs@ICG). As can be seen from Figure 6, compared with the PBS-treated group without ROS (i.e., -ROS), in the presence of ROS (i.e., +ROS), the nanoparticle drug preparation sheds from the cell surface within 12 hours, and the shedding efficiency exceeds 80%, demonstrating that the system has good ROS-responsive release ability.

[0119] 6.NK Cell-RVG-NPs@F-DMC significantly improved motor behavior disorders and dopamine neuron damage in AAV-A53Tα-Syn model mice

[0120] A PD mouse model was prepared using AAV-A53Tα-Syn, and the drug was injected into the tail vein for treatment. The experiment was divided into 5 groups, namely the control group was given PBS (wild type, abbreviated as WT), a model group was given PBS (AAV-A53Tα-Syn), a model group was given NK cells (AAV-A53Tα-Syn+NK Cell), a model group was given NPs@F-DMC prepared in Example 1 (AAV-A53Tα-Syn+NPs@F-DMC), and a model group was given the NK nano drug preparation prepared in Example 1 (AAV-A53Tα-Syn+NK Cell-RVG-NPs@F-DMC). Then, an open field test, a climbing pole test, a roller test, and a hanging test were performed. The results are shown in Figure 7.

[0121] Figures 7 and 8 show the results of the NK nanodrug preparation of Example 1 treating and improving the motor behavior disorders and dopamine neuron damage in AAV-A53Tα-Syn model mice; as can be seen from Figure 7, the NK Cell-RVG-NPs@F-DMC experimental group significantly increased the movement speed of mice in the open field test (see Figure 7A).

[0122] In addition, after treatment, the mice's climbing time was significantly reduced (see Figure 7B), and the roller movement time and hanging time were significantly prolonged (see Figure 7C and D). These behavioral experiments confirmed that the motor behavior disorders of PD model mice were significantly alleviated. The effect on dopamine neurons was detected by immunohistochemical staining. The results showed that compared with other groups, the number of dopamine neurons increased significantly after NK Cell-RVG-NPs@F-DMC treatment, indicating that F-DMC can significantly improve neuronal activity in PD models (see Figure 8).

[0123] 7.NK Cell-RVG-NPs@F-DMC significantly improved the pathological α-synuclein expression in AAV-A53T α-Syn model mice

[0124] Immunoblotting experiments further confirmed that the NK nanoparticle drug preparation of Example 1 significantly improved the pathological α-synuclein expression in AAV-A53T α-Syn model mice, as shown in FIG9 .

[0125] FIG9 shows that the NK nanomedicine preparation of Example 1 reduces the pathological α-synuclein expression in AAV-A53Tα-Syn model mice.

[0126] As can be seen from Figure 9, compared with the model group (AAV-A53Tα-Syn group), NK Cell-RVG-NPs@F-DMC treatment can significantly increase TH expression and reduce phosphorylated α-synuclein expression, showing the protective effect of the NK Cell-RVG-NPs@F-DMC delivery system on dopamine neurons (see Figure 9, Panel A). By immunofluorescence, it was found that the expression of pathological α-synuclein can be significantly reduced after NK Cell-RVG-NPs@F-DMC treatment (see Figure 9, Panel B, the arrow indicates phosphorylated α-synuclein). This confirms that the NK Cell-RVG-NPs@F-DMC delivery system can significantly eliminate pathological α-synuclein, thereby exerting a neuroprotective effect on the PD model.

Claims

1. NK nano drug preparation, characterized in that, comprising NK cells and C grafted on the surface of the NK cells 17 H 15 FO3.

2. The NK nano drug preparation according to claim 1, characterized in that: The NK nano drug preparation further comprises polylactic acid-glycolic acid copolymer and / or active oxygen responsive phospholipids, wherein the C 17 H 15 FO3 is coated with polylactic acid-glycolic acid copolymer and / or active oxygen responsive phospholipids and then grafted with the NK cells.

3. The NK nano drug preparation according to claim 2, characterized in that: The weight average molecular weight of the polylactic acid-glycolic acid copolymer is 30k-60k.

4. The NK nano drug preparation according to claim 2, characterized in that: The active oxygen responsive phospholipid is distearoyl phosphatidylethanolamine-polyethylene glycol.

5. A method for preparing a NK nano drug preparation, characterized in that: The following steps are involved: (1) Take C 17 H 15 FO3, poly(lactic-co-glycolic acid) and / or active oxygen responsive phospholipids are mixed with an organic solvent, ultrasonically dispersed, added with water, and dialyzed to prepare NPs@F-DMC; (2) mixing the targeting peptide RVG29 with the NPs@F-DMC prepared in step (1) to prepare RVG-NPs@F-DMC; (3) adding the NK cells to a phosphate buffered saline solution containing tris(2-carboxyethyl)-phosphine hydrochloride, incubating, and washing to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride, and then mixing the RVG-NPs@F-DMC with the NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride, incubating, and preparing a NK nano drug preparation.

6. The preparation method according to claim 5, characterized in that: In step (1), the C 17 H 15 The mass ratio of FO3, polylactic acid-glycolic acid copolymer, and active oxygen responsive phospholipid is 1:(0.8-3):(0.8-3).

7. The preparation method according to claim 5, characterized in that: In step (2), 1-5 μg of the targeting peptide RVG29 is mixed with the NPs@F-DMC prepared in step (1) for 0.5-4 hours, and ultrafiltration is performed to remove free targeting peptide RVG29 to obtain the RVG-NPs@F-DMC.

8. The preparation method according to claim 5, characterized in that: In step (3), the concentration of tris(2-carboxyethyl)phosphine hydrochloride in the phosphate buffered saline solution containing tris(2-carboxyethyl)phosphine hydrochloride is 0.1-1 mg / mL, and the volume of the phosphate buffered saline solution is 1-15 mL.

9. Use of the NK nano drug preparation according to any one of claims 1 to 4 in the preparation of drugs for treating neurodegenerative diseases.

10. A drug for treating Parkinson's disease, characterized in that: Including the NK nano drug preparation according to any one of claims 1-4.

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