NK nanomedicine formulations, methods of making and uses thereof

Abstract

This invention belongs to the field of pharmaceutical technology and discloses NK nanoparticle drug formulations, their preparation methods, and applications. The NK nanoparticle drug formulation includes NK cells and C0 grafted onto the surface of the NK cells. 17 H 15 FO3. In the NK nanomedicine formulation of this invention, natural killer cells and F-DMC are grafted together to facilitate the smooth crossing of the blood-brain barrier by F-DMC. Under conditions of oxidative stress damage in the brain, the connection between NK cells and F-DMC is severed. The released NK cells can clear extracellular α-synuclein, while F-DMC taken up by dopamine neurons can clear intracellular α-synuclein by promoting autophagy. Through this dual action, the pathological α-synuclein is cleared and degraded, thereby improving behavioral disorders and pathological brain damage in PD models.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to NK nanomedicine formulations, their preparation methods, and applications. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. Clinical symptoms mainly include resting tremor, bradykinesia, increased muscle tone, and postural instability. With the aging population, the incidence of PD is increasing year by year. Epidemiological data shows that the prevalence of the disease in people over 65 years of age is 1.67%. The main pathological changes in PD are the progressive degeneration and death of dopaminergic (DA) neurons in the substantia nigra of the midbrain, with the appearance of eosinophilic protein inclusion bodies formed by α-synuclein aggregation in the remaining neurons. The etiology of PD is not yet fully understood, but most scholars believe that PD is a disease caused by multiple factors, including genetic and environmental factors. To date, abnormal expression of various genes has been found to be associated with Parkinson's disease (PD). Among them, 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-synucleintrains 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 against DA neuronal damage in PD (see references: (1) 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. (2) 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. (3) 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 improvement of α-synuclein pathology by these drugs is unclear, and the safety and efficacy of such drugs in the treatment of PD remain to be discussed.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: (1) 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. (2) 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. ProcNatl Acad Sci US A. 2017; 114:E1009-E17. (3) Zhou ZD, Xie SP, Saw WT, Ho PGH, WangH, Lei Z, et al. The Therapeutic Implications of Tea Polyphenols AgainstDopamine (DA) Neuron Degeneration in Parkinson's Disease (PD). Cells. 2019; 8.).In 2019, Madeo Frank's team first reported that 4,4'-dimethoxychalcone (DMC), a flavonoid natural drug derived from Ashitaba, can activate GATA transcription factors independently of the mTORC1 signaling pathway, promoting autophagy levels in various species, including yeast, nematodes, fruit flies, mice, and primates, thereby exerting a protective effect (see: 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.). Ashitaba is abundant in Japan, and its efficacy and indications are recorded in detail in Li Shizhen's "Compendium of Materia Medica" from the Ming Dynasty and the Japanese "Yamato Honcho". Modern pharmacological studies have found that Ashitaba contains various active ingredients such as flavonoids, choline, and coumarins, with chalcones being the most prominent. Chalcones are a type of flavonoid compound with significant antioxidant activity. DMC significantly promotes autophagy and has good safety; however, its bioavailability is low, its targeting is weak, and its pathological effect on α-synuclein in PD 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 intracellular and extracellular α-synuclein, and improve behavioral disorders and brain pathological damage in Parkinson's disease models. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes NK nanomedicine formulations, their preparation methods, and applications. The preparation method of the NK nanomedicine formulations of this invention is simple and rapid, and the prepared NK nanomedicine formulations are stable and highly safe, improving the problems of low permeability of the blood-brain barrier (DMC, 4,4'-dimethoxychalcone), poor targeting, and insignificant pathological effects on α-synuclein in Parkinson's disease. In the application of the NK nanomedicine formulations of this invention in the treatment of Parkinson's disease, after crossing the blood-brain barrier, under the oxidative stress damage environment in the brain, the NK cells and targeted small molecule drugs in the NK nanomedicine formulations are released separately. By clearing intracellular and extracellular α-synuclein, it improves the motor behavior disorders in Parkinson's disease mice, and can be used as a novel neuroprotective drug for the treatment of neurodegenerative diseases.

[0005] The inventive concept of this invention is as follows: the NK nanomedicine formulation of this invention contains a lead compound (compound name: 2-Propen-1-one, 2-fluoro-1,3-bis(4-methoxyphenyl)-,(E)-,C) with significant phagocytic effect, good α-synuclein scavenging effect, and no significant toxicity) 17 H 15 FO3, with a molecular weight of 286.30, is named F-DMC. Pharmacological activity studies of F-DMC have confirmed its significant anti-PD effect. Combined with cell therapy, natural killer (NK) cells are grafted onto F-DMC, promoting its smooth crossing of the blood-brain barrier. Under intracranial oxidative stress, the connection between NK cells and F-DMC is severed. The released NK cells can clear extracellular α-synuclein, while F-DMC, taken up by dopamine neurons, can clear intracellular α-synuclein by promoting autophagy. Through this dual action, pathological α-synuclein is cleared and degraded, thereby improving behavioral disorders and intracranial pathological damage in the PD model. This treatment modality holds promise for providing new insights into the treatment of neurodegenerative diseases.

[0006] A first aspect of the present invention provides NK nanomedicine formulations.

[0007] Specifically, NK nanomedicine formulations include NK cells and C2C grafts on the surface of the NK cells. 17 H 15 FO3.

[0008] The C 17 H 15 The structural formula of FO3 is

[0009] Preferably, the NK nanomedicine formulation further includes polylactic acid-glycolic acid copolymer and / or reactive oxygen species-responsive phospholipids. The C 17 H 15 FO3 was coated with polylactic-co-glycolic acid copolymer and / or reactive oxygen species responsive phospholipids and then grafted onto NK cells.

[0010] Preferably, the polylactic acid-glycolic acid copolymer has a weight-average molecular weight of 30k-60k, more preferably 45-55k, and even more preferably 48-50k.

[0011] Preferably, in the polylactic acid-glycolic acid copolymer, the molar ratio of polylactic acid structure to glycolic acid structure is 1:(0.8-1.2), more preferably 1:1.

[0012] Preferably, the reactive oxygen species responsive phospholipid (ROS-responsive phospholipid) is distearate phosphatidylethanolamine-polyethylene glycol.

[0013] Preferably, the polyethylene glycol in the distearate phosphatidylethanolamine-polyethylene glycol is at least one of polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 6000, with polyethylene glycol 2000 being the most preferred. The phosphatidylethanolamine-polyethylene glycol is a reactive oxygen species-responsive phospholipid.

[0014] A second aspect of the present invention provides a method for preparing NK nanomedicine formulations.

[0015] Specifically, the preparation method of NK nanomedicine formulations includes the following steps:

[0016] (1) Take C 17 H 15 FO3, polylactic acid-glycolic acid copolymer, reactive oxygen species responsive phospholipids and organic solvents were mixed, ultrasonically dispersed, water was added, and dialyzed to obtain NPs@F-DMC;

[0017] (2) Mix the targeting peptide RVG29 with the NPs@F-DMC to obtain RVG-NPs@F-DMC;

[0018] (3) NK cells were added to a phosphate buffer solution containing tris(2-carboxyethyl)-phosphine hydrochloride, incubated, and washed to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride. Then, the RVG-NPs@F-DMC was mixed with the NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride and incubated to prepare an NK nanomedicine preparation (denoted as NK cell-NPs@F-DMC).

[0019] Preferably, in step (1), the C 17 H 15 The mass ratio of FO3, polylactic acid-glycolic acid copolymer, and reactive oxygen species responsive phospholipid is 1:(0.8-3):(0.8-3), preferably 1:(1-2):(1-2).

[0020] Preferably, in step (1), the organic solvent includes 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.

[0021] Preferably, in step (1), the C 17 H 15 The mass-to-volume ratio of FO3 to organic solvent is 1 mg:(0.1-1) mL, preferably 1 mg:(0.1-0.5) mL.

[0022] Preferably, in step (1), the power of the ultrasound is 20-50W, more preferably 30-35W.

[0023] Preferably, in step (1), the water is ultrapure water.

[0024] Preferably, in step (1), water is added dropwise during ultrasonic dispersion.

[0025] Preferably, in step (1), the volume of water is 1-20 mL, more preferably 2-10 mL.

[0026] Preferably, in step (1), the dialysis is performed using a dialysis bag and ultrapure water is used for dialysis for 40-48 hours.

[0027] More preferably, the dialysis bag has a molecular weight cutoff of 3000-3500 Da. The purpose of dialysis is to remove carbon dioxide. 17 H 15 FO3 and organic solvents.

[0028] 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, ultrafiltered to remove the free targeting peptide RVG29, and the RVG-NPs@F-DMC is obtained and kept at 4°C for later use.

[0029] Preferably, in step (3), the concentration of tris(2-carboxyethyl)-phosphine hydrochloride in the phosphate buffer solution containing tris(2-carboxyethyl)-phosphine hydrochloride is 0.1-1 mg / mL, preferably 0.1-0.5 mg / mL.

[0030] Preferably, in step (3), the volume of the phosphate buffer solution containing tris(2-carboxyethyl)-phosphine hydrochloride is 1-15 mL, preferably 1-10 mL.

[0031] Preferably, in step (3), the quantity is (1-10)×10 6 One NK cell was added to a phosphate buffer solution containing tris(2-carboxyethyl)-phosphonic acid hydrochloride.

[0032] Preferably, in step (3), the incubation temperature is 35-37°C and the incubation time is 5-50 minutes, with the preferred incubation temperature being 37°C and the incubation time being 10-45 minutes. After incubation, the sample is washed 2-10 times with phosphate buffer solution.

[0033] 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.

[0034] Preferably, in step (3), before obtaining the NK nanomedicine formulation, the nanomedicine is washed 2-10 times with phosphate buffer solution.

[0035] Preferably, in step (3), after incubation, the mixture is centrifuged 2-10 times and dispersed with phosphate buffer solution to remove impurities such as free RVG29 and tris(2-carboxyethyl)-phosphine hydrochloride, thereby obtaining NK nanomedicine formulation.

[0036] Preferably, the preparation method of NK nanomedicine formulation includes the following steps:

[0037] (1) Take C 17 H 15 FO3, polylactic acid-glycolic acid copolymer, reactive oxygen species-responsive phospholipids, and organic solvents were mixed, ultrasonically dispersed, water was added, and dialyzed to prepare NPs@F-DMC, C 17 H 15 The mass ratio of FO3, polylactic acid-glycolic acid copolymer, and reactive oxygen species responsive phospholipids is 1:(0.8-3):(0.8-3), C 17 H 15 The mass-to-volume ratio of FO3 to organic solvent is 1 mg:(0.1-1) mL. Dialysis is performed using a dialysis bag and ultrapure water for 40-48 hours. The molecular weight cutoff of the dialysis bag is 3500 Da, and the ultrasonic power is 20-50 W.

[0038] (2) Take 1-5 μg of the targeting peptide RVG29 and mix it with the NPs@F-DMC prepared in step (1) for 0.5-4 hours, then ultrafilter to obtain RVG-NPs@F-DMC;

[0039] (3) The quantity is (1-10)×10 6NK cells were added to a phosphate-buffered saline solution containing tris(2-carboxyethyl)-phosphonic acid hydrochloride and incubated at 35-37°C for 5-50 minutes. The cells were washed 2-10 times with the phosphate-buffered saline solution to obtain NK cells treated with tris(2-carboxyethyl)-phosphonic acid hydrochloride. Then, 1 mL of RVG-NPs@F-DMC was mixed with the NK cells treated with tris(2-carboxyethyl)-phosphonic acid hydrochloride and incubated at 35-37°C for 5-50 minutes. After centrifugation, the cells were washed 2-10 times with the phosphate-buffered saline solution to obtain the NK nanomedicine formulation (denoted as NKcell-NPs@F-DMC). The concentration of tris(2-carboxyethyl)-phosphonic acid hydrochloride in the phosphate-buffered saline solution was 0.1-1 mg / mL, and the volume of the phosphate-buffered saline solution was 1-15 mL.

[0040] A third aspect of the present invention provides the application of NK nanomedicine formulations.

[0041] The above-mentioned NK nanomedicine formulations are used in the preparation of drugs for treating neurodegenerative diseases.

[0042] Preferably, the neurodegenerative disease includes Parkinson's disease.

[0043] A drug for treating Parkinson's disease includes the aforementioned NK nanomedicine formulation.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] In this invention, the NK nanomedicine formulation grafts natural killer cells (NK cells) and F-DMCs, facilitating the smooth crossing of the blood-brain barrier by F-DMCs. Under conditions of oxidative stress damage in the brain, the connection between NK cells and F-DMCs is severed. The released NK cells can clear extracellular α-synuclein, while F-DMCs taken up by dopamine neurons can clear intracellular α-synuclein by promoting autophagy. Through this dual action, the pathological α-synuclein is cleared and degraded, thereby improving behavioral disorders and pathological brain damage in PD models. This treatment modality holds promise for providing new insights into the treatment of neurodegenerative diseases. Attached Figure Description

[0046] Figure 1 Here are structural diagrams of F-DMC and DMC;

[0047] Figure 2 A graph showing the level of autophagy significantly promoted by F-DMC in dopaminergic neurons MN9D cells;

[0048] Figure 3 The diagram shows the effect of F-DMC in significantly promoting the clearance of pathological α-synuclein by dopaminergic neurons MN9D cells;

[0049] Figure 4 The particle size distribution results are for NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1.

[0050] Figure 5 Scanning electron microscope images of NK cells and the NK nanomedicine formulation prepared in Example 1;

[0051] Figure 6 Results of ROS-responsive drug release performance assay for labeled NK nanomedicine formulations (NK cell-NPs@ICG);

[0052] Figure 7 The NK nanomedicine formulation of Example 1 improved the motor behavior disorder in AAV-A53Tα-Syn model mice.

[0053] Figure 8 The NK nanomedicine formulation of Example 1 improved the dopamine neuron damage in AAV-A53Tα-Syn model mice;

[0054] Figure 9 The NK nanomedicine formulation of Example 1 reduced the pathological α-synuclein expression in AAV-A53Tα-Syn model mice. Detailed Implementation

[0055] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0056] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0057] Example 1: Preparation of NK nanomedicine formulations

[0058] The preparation method of NK nanomedicine formulation includes the following steps:

[0059] (1) Take 5mg C 17 H 15FO3 (F-DMC, purchased from ChemDiv, USA), 10 mg of polylactic acid-glycolic acid copolymer (the weight-average molecular weight of the polylactic acid-glycolic acid copolymer was 50 kDa, and the molar ratio of polylactic acid structure to glycolic acid structure in the polylactic acid-glycolic acid copolymer was 1:1), 10 mg of distearylphosphatidylethanolamine-polyethylene glycol (the polyethylene glycol in distearylphosphatidylethanolamine-polyethylene glycol was polyethylene glycol 2000) and 1 mL of organic solvent (the organic solvent was a mixture of ethanol and dimethyl sulfoxide, and the volume ratio of ethanol to dimethyl sulfoxide was 1:9) were mixed and dispersed dropwise with 5 mL of ultrapure water under ultrasonic conditions at a power of 30 W. Then, the mixture was dialyzed (dialysis was performed using a dialysis bag and ultrapure water for 48 hours, and the molecular weight cutoff of the dialysis bag was 3500 Da) to obtain NPs@F-DMC.

[0060] (2) Take 2 μg of the target peptide RVG29 and mix it with the NPs@F-DMC prepared in step (1) for 2 hours, and ultrafilter the free target peptide RVG29 to obtain RVG-NPs@F-DMC;

[0061] (3) The quantity is 5×10 6 NK cells were added to phosphate-buffered saline (PBS) containing tris(2-carboxyethyl)-phosphonic acid hydrochloride (0.3 mg / mL, 5 mL). The cells were incubated at 37°C for 20 minutes and washed three times with PBS to obtain NK cells treated with tris(2-carboxyethyl)-phosphonic acid hydrochloride. Then, 1 mL of RVG-NPs@F-DMC (20 μg / mL) was mixed with 1×10⁻⁶ PBS. 5 NK cells treated with tris(2-carboxyethyl)-phosphonic acid hydrochloride were mixed and incubated at 37°C for 30 minutes. The mixture was then washed three times with phosphate-buffered saline solution to obtain the NK nanomedicine formulation (denoted as NK cell-NPs@F-DMC).

[0062] Example 2: Preparation of NK nanomedicine formulations

[0063] The preparation method of NK nanomedicine formulation includes the following steps:

[0064] (1) Take 5mg C 17 H 15FO3 (F-DMC), 5 mg of polylactic acid-glycolic acid copolymer (the weight-average molecular weight of the polylactic acid-glycolic acid copolymer is 50 kDa, and the molar ratio of polylactic acid structure to glycolic acid structure in the polylactic acid-glycolic acid copolymer is 1:1), 5 mg of distearylphosphatidylethanolamine-polyethylene glycol (the polyethylene glycol in distearylphosphatidylethanolamine-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 dispersed by adding 10 mL of ultrapure water dropwise under ultrasonic conditions with a power of 30 W. Then, the mixture was dialyzed (dialysis was performed using a dialysis bag and ultrapure water for 48 hours, and the molecular weight cutoff of the dialysis bag was 3500 Da) to obtain NPs@F-DMC.

[0065] (2) Take 2 μg of the target peptide RVG29 and mix it with the NPs@F-DMC prepared in step (1) for 1 hour, and ultrafilter the free target peptide RVG29 to obtain RVG-NPs@F-DMC;

[0066] (3) The quantity is 2×10 6 NK cells were added to a phosphate-buffered saline solution containing tris(2-carboxyethyl)-phosphonic acid hydrochloride (tris(2-carboxyethyl)-phosphonic acid hydrochloride concentration of 0.1 mg / mL, phosphate-buffered saline solution volume of 3 mL), incubated at 37°C for 45 minutes, and washed three times with phosphate-buffered saline solution to obtain NK cells treated with tris(2-carboxyethyl)-phosphonic acid 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)-phosphonic acid hydrochloride were mixed and incubated at 37°C for 30 minutes. The mixture was then washed three times with phosphate-buffered saline solution to obtain the NK nanomedicine formulation (denoted as NK cell-NPs@F-DMC).

[0067] The product effectiveness testing methods are as follows:

[0068] 1. ROS-responsive drug release experiment of NK nanoparticle drug formulation

[0069] Labeled nanomedicine formulations (denoted as RVG-NPs@ICG) were prepared by replacing F-DMC with 2% (w / w) of the near-infrared fluorescent dye ICG, and further labeled NK nanomedicine formulations (denoted as NK cell-NPs@ICG) were prepared. The labeled NK nanomedicine formulations (NK cell-NPs@ICG) were co-incubated at 37°C with ROS-containing culture medium (400 mM H2O2 and 3.2 μM copper chloride) and ROS-free culture medium. At different time points, the mixture was centrifuged at low speed (300 g, 3 min), and a certain volume of the supernatant was taken to measure its fluorescence intensity. The same volume of culture medium was added to the co-incubation dishes. The release amount of NPs@ICG at different time points was calculated using the following formula. The results are shown below. Figure 6 As shown.

[0070] NPs@ICG cumulative release rate

[0071] In the formula: V e For the displacement volume, V o F is the volume of the released liquid. i : represents the fluorescence intensity in the released liquid during the i-th replacement sampling.

[0072] 2. Cell viability assay

[0073] (1) Add 100 μL of cell suspension to a 96-well plate, with 5000 cells per well.

[0074] (2) After culturing the cells in a cell culture incubator for 24 hours, the cells were subjected to experimental treatment.

[0075] (3) After the time is reached, discard the supernatant, prepare the working solution by mixing the CCK-8 detection reagent and the 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.

[0076] (4) After incubation for 1 hour, the absorbance was measured at a wavelength of 450 nm.

[0077] 3. Electron microscopy examination

[0078] The experimental steps for transmission electron microscopy (TEM) detection are as follows:

[0079] (1) Fix with 2.5% glutaraldehyde in 0.1M PBS fixative at room temperature for 1 hour, then rinse 3 times with 0.1M PBS.

[0080] (2) Stain with 1% osmium tetroxide for 30 minutes, rinse three times with distilled water for 15 minutes each time.

[0081] (3) Dehydrate with 50%, 70%, 90% and 100% alcohol at 4°C in sequence, for 15-20 minutes for each gradient.

[0082] (4) Acetone / embedding agent (1:1), room temperature for 1 hour; acetone / embedding agent (1:3), overnight at 4°C; pure embedding agent, overnight at 4°C.

[0083] (5) The resin-impregnated sample was cured in an oven at 65°C for 24 hours.

[0084] (6) Slice the material using an ultramicro slicer with a thickness of 50-100 nm.

[0085] (7) 3% uranium acetate-lead citrate double staining.

[0086] (8) Observe and photograph using transmission electron microscopy.

[0087] 4. PD model preparation and drug treatment

[0088] The PD model was developed using brain-targeted injection of AAV-A53Tα-Syn virus. The brain-targeted injection procedure was as follows: After anesthetizing C57BL / 6J mice with isoflurane, they were fixed in a prone position on a stereotaxic instrument, with the incisors 2.3 mm below the ear rods, ensuring the anterior and posterior fontanelles were on the same horizontal plane. The hair on the head was shaved, the scalp was disinfected with iodine, and the scalp was cut open and the periosteum dissected. The substantia nigra coordinates were: 3.0 mm posterior to the anterior fontanelle, 1.3 mm lateral to the midline, and 4.7 mm subperiosteally. 0.5 μL of either the control virus or AAV-A53Tα-Syn virus was injected. After injection, the injection was stopped for 10 minutes, the needle was slowly withdrawn, a stitch was placed, and the mouse was kept warm. Penicillin was administered intramuscularly. After waking up, the mice were placed in cages for feeding. One month after viral injection, PBS, NK cells, and nano-encapsulated F-DMC (NPs@F-DMC) were administered via tail vein, with NK cells linked to F-DMC (NK Cell-NPs@F-DMC), once every other day for a total of 8 treatments. Behavioral and other assessments were performed after the final treatment.

[0089] 5. Behavioral experiments

[0090] (1) Open field experiment: The open field box was a square open box with a length of 50cm, a width of 50cm, and a height of 40cm, and the inner walls were painted black. The top of the open box was artificially illuminated. The open box was divided into a central area (25cm long and 25cm wide) and an outer peripheral area. At the beginning of the experiment, the mice were placed in the center of the open box, and each mouse was kept in the same position and orientation. The mice were allowed to move freely in the open box for 15 minutes. The movement speed of the mice was recorded using Smart 3.0 video tracking software.

[0091] (2) Roller test: The roller test requires the animal to maintain balance and move continuously on the roller. It is a widely used test to detect motor coordination. The roller diameter is 6cm and the rotation speed is 20r / min. After the mouse adapts 5 times, the interval between each test is 1min. The average value is taken after 5 consecutive tests.

[0092] (3) Pole Climbing Experiment: A small foam ball with a diameter of 2 cm was fixed to the top of a 50 cm long and 1 cm 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 its hind limbs on the ball). It was allowed to climb down naturally. The length of the climb from when the mouse stood on the top of the pole to when its forelimbs touched the bottom platform was considered the complete climb. After modeling, a certain number of mice from each group were selected for the pole climbing experiment. The behavioral changes of the mice were observed, and the climbing time was recorded.

[0093] (4) Suspension Experiment: A homemade acrylic test box was used. A 1.5 mm diameter metal rod was placed horizontally 30 cm above the ground. A cover was placed 1 cm above the metal rod to prevent the mice from rolling over onto it. During the experiment, the mice were suspended from the metal rod with their front paws gripping it for 10 seconds. The suspension scores were calculated as follows: 3 points for mice gripping the rod with both hind paws; 2 points for mice gripping the rod with one hind paw; 1 point for mice failing to grip the rod with both hind paws; and 0 points for mice that fell.

[0094] 6. Immunoblot hybridization experiment

[0095] Protein samples were extracted from cells, and protein concentration was determined using a BCA kit. Loading buffer was added at a specific ratio, and the mixture was incubated in a boiling water bath at 100°C for 5 minutes. After electrophoresis, the proteins were transferred to a PVDF (polyvinylidene fluoride) membrane. At room temperature, the PVDF membrane was washed with TBST buffer (Tris salt buffer) for 5 minutes, blocked with 5% bovine serum albumin blocking solution for 2 hours, and washed three times with TBST buffer for 5 minutes each time. The membrane was incubated overnight at 4°C with primary antibody. The membrane was then washed three times with TBST buffer for 5 minutes each time, and then incubated with secondary antibody at room temperature for 1 hour, followed by washing with TBST buffer. Equal volumes of chemiluminescent reagent A and B were mixed, and the PVDF membrane was incubated for 1 minute before exposure.

[0096] 7. Immunohistochemical staining

[0097] Mice were anesthetized with isoflurane and perfused with pre-cooled saline and 4% paraformaldehyde. Mouse brains were fixed in 4% paraformaldehyde at 4°C for approximately 6 hours, then sequentially transferred to 20% and 30% sucrose solutions for graded dehydration at 4°C. Tissue was embedded in OCT embedding medium and serially sectioned using a cryostat. Sections were placed in a 0.01M citrate buffer staining tank and incubated at 98°C for 10–15 minutes, then allowed to cool naturally to room temperature. The sections were washed three times with PBS for 5 minutes each time. The sections were then blocked with 0.3% Triton and 5% BSA for 30 minutes, followed by three washes with PBS for 5 minutes each time. Primary antibody was added, and the sections were incubated overnight at 4°C in a humidified chamber. The next day, the sections were removed from the freezer and warmed to room temperature for 45 minutes. Secondary antibody was added, and the sections were incubated at 37°C for 60 minutes. The sections were washed three times with PBS for 5 minutes each time. DAB chromogenic solution was added to the sections, and the reaction time was monitored under a microscope at room temperature. The sections were then rinsed with running tap water. Counterstain with hematoxylin for 2 minutes, separate the colors once with hydrochloric acid and alcohol, and rinse with running tap water. After dehydration with graded alcohols, mount with resin and examine under a microscope.

[0098] 8. Immunofluorescence staining

[0099] After rinsing with PBS, the slides were blocked with PBS containing 0.3% Triton and 5% BSA at 37°C for 1 hour, followed by incubation with primary antibody at 4°C overnight. After washing three times with PBS, the slides were incubated with secondary antibody for 1 hour, air-dried, and mounted with glycerol. The slides were then scanned under a laser confocal microscope, and the data was acquired and digitally imaged using a computer.

[0100] The product effectiveness test results are as follows:

[0101] 1. Cellular experimental verification of F-DMC promoting autophagy

[0102] The dopamine neuron cell line MN9D was cultured and treated with different 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 results were observed by Western blotting and electron microscopy. Figure 2 ( Figure 2 In the diagram, “Ctrl” represents the rapamycin positive control, and LC3Ⅰ, LC3Ⅱ, p62, and GAPDH represent proteins.

[0103] Figure 1 Here are structural diagrams of F-DMC and DMC; Figure 1 Figure A in the diagram represents the two-dimensional structure of DMC, and Figure B represents the two-dimensional structure of F-DMC.

[0104] Figure 2 A graph showing the level of autophagy in F-DMC-mediated dopaminergic neurons MN9D. From... Figure 2It can be seen that F-DMC can promote autophagy in MN9D cells in a concentration-dependent manner, specifically by increasing the LC3Ⅱ / Ⅰ ratio (see [link to relevant documentation]). Figure 2 (See Figure A in the table). Furthermore, at the same concentration (100 μM), F-DMC significantly promoted autophagy compared to DMC (see Figure A in the table). Figure 2 (See Figure B in the original text). Transmission electron microscopy revealed that F-DMC significantly promoted the formation of autolysosomes, specifically manifested as an increase in the number of lysosomes (see Figure B in the original text). Figure 2 (See Figure C in the diagram). Cell viability assays showed that different concentrations of F-DMC did not exhibit cytotoxicity, confirming the safety of the drug (see Figure C in the diagram). Figure 2 (D diagram in the image).

[0105] 2. F-DMC significantly promotes the clearance and degradation of pathological α-synuclein.

[0106] MN9D cells were treated with human α-synuclein fibroblasts (hα-Syn), and F-DMC and DMC (100 μM) were added. After culturing for 24 hours, the results were as follows: Figure 3 As shown.

[0107] Figure 3 ( Figure 3 The “Ctrl” in the image represents the rapamycin positive control. This image shows the effect of F-DMC in significantly promoting the clearance of pathological α-synuclein by dopaminergic neurons MN9D cells. Figure 3 It can be seen that, compared with DMC, F-DMC can significantly clear α-synuclein fibrils (manifested as reduced expression of phosphorylated α-synuclein) (see... Figure 3 (See Figure A in the table). Furthermore, 50 and 100 μM F-DMC significantly scavenged phosphorylated α-synuclein and reduced endogenous α-synuclein expression, demonstrating a good effect in degrading α-synuclein (see Figure A in the table). Figure 3 (Figure B in the diagram).

[0108] 3. Characterization of intermediates in NK nanomedicine formulations

[0109] The particle size of NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1 was detected using a Zetasizer Nano ZS instrument, and the results are as follows: Figure 4 As shown.

[0110] Figure 4 The particle size distribution results are shown for NPs@F-DMC and RVG-NPs@F-DMC prepared in Example 1. Figure 4 ( Figure 4In this context, "Size" refers to particle size, and "PDI" refers to polydispersity index. It can be seen that the particle size distribution of NPs@F-DMC and RVG-NPs@F-DMC is between 80-300 nm (see [link to documentation]). Figure 4 Figure A in the figure shows that the average particle size is between 160.0 ± 6.9 nm and 167.3 ± 6.5 nm (see Figure A in the figure). Figure 4 (See Figure B in the table). The polydispersity indices of PDI are 0.445 and 0.403, respectively (see Figure B in the table). Figure 4 (Figure C in the diagram).

[0111] 4. Scanning electron microscopy characterization results of NK nanomedicine formulations

[0112] Figure 5 Scanning electron microscope images of NK cells and the NK nanomedicine formulation prepared in Example 1; Figure 5 In this context, "NK cell" refers to NK cells, and "NK cell-NPs@F-DMC" refers to the NK nanoparticle drug formulation prepared in Example 1, where the drug grafted onto the surface of NK cells is as follows: Figure 5 As indicated by the arrow.

[0113] 5. ROS-responsive drug release experiment of NK nanoparticle drug formulation

[0114] A fluorescently labeled nanomedicine formulation (denoted as RVG-NPs@ICG) was prepared by replacing F-DMC with near-infrared fluorescent dye ICG at a mass ratio of 2%, following the method in Example 1. A fluorescently labeled NK nanomedicine formulation (denoted as NKcell-NPs@ICG) was further prepared. The fluorescently labeled NK nanomedicine formulation (NK cell-NPs@ICG) was co-incubated at 37°C with ROS-containing culture medium (400 mM H2O2 and 3.2 μM copper chloride) and ROS-free culture medium. At different time points, the mixture was centrifuged at low speed (300 g, 3 minutes), and a certain volume of the supernatant was taken to measure its fluorescence intensity. The same volume of culture medium was added to the co-incubation dishes. The cumulative release rate of NPs@ICG at different time points was calculated using the following formula. The results are shown below. Figure 6 As shown.

[0115] NPs@ICG cumulative release rate

[0116] In the formula: V e For the displacement volume, V o F is the volume of the released liquid. i : represents the fluorescence intensity in the released liquid during the i-th replacement sampling.

[0117] Figure 6The results show the ROS-responsive drug release performance of fluorescently labeled NK nanoparticles (NK cell-NPs@ICG); from Figure 6 As can be seen, compared with the PBS treatment group without ROS (i.e. -ROS), the nanomedicine formulation detached from the cell surface at an efficiency of over 80% 12 hours in the presence of ROS (i.e. +ROS), demonstrating that the system has good ROS-responsive release capability.

[0118] 6. NK Cell-RVG-NPs@F-DMC significantly improved motor behavioral disorders and dopamine neuron damage in AAV-A53Tα-Syn model mice.

[0119] A PD mouse model was established using AAV-A53Tα-Syn, and the drug was administered via tail vein injection. The experiment was divided into five groups: a control group receiving PBS (wild-type, WT); one model group receiving PBS (AAV-A53Tα-Syn); one model group receiving NK cells (AAV-A53Tα-Syn + NK Cell); one model group receiving NPs@F-DMC prepared in Example 1 (AAV-A53Tα-Syn + NPs@F-DMC); and one model group receiving the NK nanoparticle drug formulation prepared in Example 1 (AAV-A53Tα-Syn + NK Cell-RVG-NPs@F-DMC). Open field, pole climbing, roller, and suspension experiments were then performed. The results are as follows: Figure 7 As shown.

[0120] Figure 7 and Figure 8 The NK nanomedicine formulation used in Example 1 improved motor behavioral disorders and dopamine neuron damage in AAV-A53Tα-Syn model mice; from Figure 7 It can be seen that the NK Cell-RVG-NPs@F-DMC experimental group significantly increased the movement speed of mice in the open field experiment (see...). Figure 7 (Figure A in the middle)

[0121] In addition, the pole-climbing time of mice was significantly reduced after treatment (see [link]). Figure 7 Figure B shows that the roller movement time and suspension time are significantly prolonged (see Figure B). Figure 7 (See Figures C and D). These behavioral experiments confirmed that motor behavioral disorders in PD model mice were significantly alleviated. Immunohistochemical staining to examine its effect on dopamine neurons showed that, compared with other groups, the number of dopamine neurons was significantly increased after NKCell-RVG-NPs@F-DMC treatment, indicating that F-DMC can significantly improve neuronal activity in the PD model (see Figure C and D). Figure 8 ).

[0122] 7. NK Cell-RVG-NPs@F-DMC significantly improved pathological α-synuclein expression in AAV-A53Tα-Syn model mice.

[0123] Further confirmation was made using immunoblotting hybridization experiments, and the results were as follows: Figure 9 As shown.

[0124] Figure 9 The NK nanomedicine formulation of Example 1 reduced the pathological α-synuclein expression in AAV-A53Tα-Syn model mice.

[0125] from Figure 9 It can be seen that, 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, demonstrating the protective effect of the NK Cell-RVG-NPs@F-DMC administration system on dopamine neurons (see [link to relevant documentation]). Figure 9 (Figure A). Immunofluorescence revealed that NK Cell-RVG-NPs@F-DMC treatment significantly reduced the expression of pathological α-synuclein (see Figure A). Figure 9 Figure B shows the phosphorylated α-synuclein (arrow indicates phosphorylated α-synuclein). This confirms that the NK Cell-RVG-NPs@F-DMC drug delivery system can significantly clear pathological α-synuclein, thereby exerting a neuroprotective effect on the PD model.

Claims

1. An NK nanomedicine formulation, characterized in that, Includes NK cells and F-DMC grafted onto the surface of the NK cells, wherein the F-DMC is grafted onto the NK cells after being coated with polylactic-co-glycolic acid copolymer and reactive oxygen species responsive phospholipids; The chemical formula of the F-DMC is C 17 H 15 FO3, structural formula is 。 2. The NK nanomedicine formulation according to claim 1, characterized in that, The polylactic acid-hydroxyacetic acid copolymer has a weight-average molecular weight of 30k-60k.

3. A method for preparing an NK nanomedicine formulation, characterized in that, Includes the following steps: (1) Take F-DMC, polylactic acid-glycolic acid copolymer, reactive oxygen species responsive phospholipids and organic solvent, mix them, disperse them by ultrasonication, add water, dialyze, and obtain NPs@F-DMC; The chemical formula of the F-DMC is C 17 H 15 FO3, structural formula is ； (2) Mix the targeting peptide RVG29 with the NPs@F-DMC prepared in step (1) to obtain RVG-NPs@F-DMC; (3) NK cells were added to a phosphate buffer solution containing tris(2-carboxyethyl)-phosphine hydrochloride, incubated, and washed to obtain NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride. Then, the RVG-NPs@F-DMC was mixed with the NK cells treated with tris(2-carboxyethyl)-phosphine hydrochloride and incubated to prepare NK nanomedicine formulation.

4. The preparation method according to claim 3, characterized in that, In step (1), the mass ratio of F-DMC, polylactic acid-glycolic acid copolymer, and reactive oxygen species responsive phospholipid is 1:(0.8-3):(0.8-3).

5. The preparation method according to claim 3, 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 ultrafiltered to remove the free targeting peptide RVG29, thereby obtaining the RVG-NPs@F-DMC.

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

7. The use of the NK nanomedicine formulation according to any one of claims 1-2 in the preparation of a medicament for treating neurodegenerative diseases.

8. A drug for treating Parkinson's disease, characterized in that, Includes the NK nanomedicine formulation according to any one of claims 1-2.

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