NK cell drug delivery system, preparation method therefor and use thereof

Abstract

An NK cell drug delivery system, a preparation method therefor and a use thereof, relating to the field of drugs. The NK cell drug delivery system comprises activated NK cells and drug-loaded nanoparticles linked to the surfaces of the activated NK cells; the activated NK cells are NK cells containing sulfhydryl on the surfaces; maleimide groups or sulfhydryl groups are contained on the surfaces of the drug-loaded nanoparticles; the drug-loaded nanoparticles are linked to sulfhydryl on the surfaces of the activated NK cells by means of the maleimide groups or the sulfhydryl groups; by means of the NK cells linked to sulfhydryl by means of the maleimide groups or the sulfhydryl groups, and the drug-loaded nanoparticles, the NK cell drug delivery system has high targeting performance and low biotoxicity on cells or tissues of brain diseases, and significantly improves the effect of treating brain diseases.

Description

A NK cell drug delivery system and its preparation method and use Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a NK cell drug delivery system and a preparation method and use thereof. Background Art

[0002] The global incidence of brain diseases is high and continues to rise, creating a massive market. However, drug development for these diseases remains challenging, characterized by high development costs, a long clinical path, and a high failure rate. The brain is highly protected by physiological barriers, particularly the blood-brain barrier (BBB), and this barrier is a major obstacle in the development of drugs for brain diseases. Over 98% of small molecule drugs have difficulty crossing the BBB and entering the central nervous system. Without active transport, drugs cannot enter the brain, resulting in a large number of in vitro effective drugs being prematurely rendered ineffective due to their inability to cross the BBB. More importantly, once drugs cross the BBB, they must be further targeted to diseased cells. Optimizing drug accumulation in diseased cells without damaging normal brain cells presents another challenge. Enabling drugs to penetrate the BBB and improve their targeting efficiency at brain lesions has become crucial for the treatment of brain diseases.

[0003] There are currently two main treatments for brain diseases: surgery and systemic or local administration of small molecule drugs. For some brain inflammatory diseases, such as brain abscesses and brain tumors, surgical treatment can achieve the therapeutic purpose by removing the lesion or draining the pus. However, surgical treatment has certain risks and may cause surgery-related complications and side effects. In addition, some brain diseases are not suitable for surgical treatment, such as meningitis, encephalitis, and neurodegenerative diseases. Small molecule drug therapy is one of the most common and conventional treatment methods. However, most drugs have difficulty effectively crossing the blood-brain barrier, which reduces the therapeutic effect of the drugs; in addition, drugs cannot directly repair damaged brain tissue and can only alleviate inflammatory symptoms; some drugs may have side effects and toxic reactions; some pathogens may develop drug resistance, resulting in reduced therapeutic effects.

[0004] In recent years, the rapid development of cell-based drug delivery systems has provided new avenues for the treatment of brain diseases. Although the blood-brain barrier (BBB) ​​and blood-cerebrospinal fluid barrier (BCSFB) strictly restrict the entry of cells and molecules into the brain parenchyma, immune cells can cross these barriers, particularly in pathological conditions such as stroke and inflammation. Furthermore, cell-based drug delivery systems offer numerous advantages as a novel therapeutic strategy. 1) Targetedness: Cell-based drug delivery systems can be designed to deliver targeted therapeutic effects, identifying and targeting diseased cells or tissues, providing more accurate and effective treatment. 2) Personalized Therapy: Because cell delivery vehicles are often derived from the patient's own cells or exogenous cells that have undergone personalized treatment, personalized treatment strategies can be implemented, taking into account the patient's unique biological characteristics and needs. 3) Overcoming Immune Escape: Cell-based drug delivery systems can overcome the immune escape challenges faced by some traditional drugs. For example, tumor cells may evade immune attack by altering surface antigen expression or other mechanisms, while cell-based drugs can recognize and attack these evading cells through a variety of mechanisms. 4) Long-lasting Effects: Cell-based drugs can survive and remain active in the body for extended periods, providing lasting therapeutic effects. In contrast, some traditional drugs may require frequent dosing to maintain their therapeutic effects. 5) Multiple therapeutic mechanisms: Cellular drugs can exert their therapeutic effects through a variety of mechanisms, such as directly killing diseased cells, secreting cytokines and intercellular signaling molecules, and modulating immune responses, thereby influencing disease progression and therapeutic outcomes at multiple levels. Designing and constructing novel cell-based drug delivery systems for brain diseases to improve their effectiveness is a technical problem and a pressing goal addressed by this invention. Technical issues

[0005] In order to overcome the technical problem that existing drugs have poor therapeutic effects on brain diseases, the present invention proposes a novel NK cell drug delivery system and its preparation method. The NK cell drug delivery system has high targeting and low biological toxicity to brain disease cells or tissues, and has a simple preparation process, which significantly improves the effect of treating brain diseases. Technical Solutions

[0006] The present invention provides an NK cell drug delivery system, which includes activated NK cells and drug-loaded nanoparticles bound to the surface of the activated NK cells, wherein the activated NK cells are NK cells containing thiol groups on the surface, and the drug-loaded nanoparticles contain maleimide groups or thiol groups on the surface of the activated NK cells through the maleimide groups or thiol groups.

[0007] Furthermore, the activated NK cells are prepared by reducing the disulfide bonds of NK cell surface proteins to sulfhydryl groups using a sulfhydryl reducing agent; optionally, one or more of tris(2-carboxyethyl)phosphine hydrochloride, mercaptoethanol, dithiothreitol, dimercaptopropanol, β-mercaptoethylamine and cysteine.

[0008] Furthermore, the drug-loaded nanoparticles include a carrier shell and a drug encapsulated in the carrier shell, the carrier shell contains a polymer carrier or albumin, wherein the polymer carrier includes a first polymer material and phospholipid-ketalthioalcohol-polyethylene glycol-maleimide, and the first polymer material is selected from at least one of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), and polycaprolactone (PCL); preferably, the mass ratio of the first polymer material to the phospholipid-ketalthioalcohol-polyethylene glycol-maleimide is 3-15:3-15; preferably, the phospholipid-ketalthioalcohol-polyethylene glycol-maleimide is distearoylphosphatidylethanolamine-ketalthioalcohol-polyethylene glycol-maleimide; preferably, the mass ratio of the drug to the polymer carrier or albumin is 3-7:2-30.

[0009] More preferably, the mass ratio of the first polymer material to phospholipid-thioketal-polyethylene glycol-maleimide is 1:1.

[0010] Preferably, the mass ratio of the drug, the first polymer material and the phospholipid-thioketal-polyethylene glycol-maleimide is 3-7:3-15:3-15, more preferably 5:10:10.

[0011] Optionally, the molecular weight of the first polymer material is 20k-100k.

[0012] Specifically, the first polymer material may be PLGA50k, PLGA20k, PLGA100k, PLA50k, PCL50k, etc.

[0013] Furthermore, the drug in the drug-loaded nanoparticles includes one or more of a simulated drug and a drug for preventing or treating brain diseases.

[0014] The simulated drugs include, but are not limited to, fluorescent dyes such as pyrene, perylene, BODIPY, fluorescein, rhodamine, cyanine dyes, ICG series dyes and their derivatives.

[0015] The brain diseases include but are not limited to Alzheimer's disease, traumatic brain injury, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, depression, neuroinflammation, cerebrovascular disease, hydrocephalus, brain tumors; brain tumors include primary gliomas, secondary tumor brain metastases, oligodendrogliomas, meningiomas, astrocytomas, meningiomas and other brain tumors.

[0016] Among them, drugs for treating Alzheimer's disease include: fingolimod, 3-n-butylphthalide and its derivatives, curcumin, sildenafil, exenatide (Exendin-4), tripterygium wilfordii, omega-3 (Omega-3) polyunsaturated fatty acids, Malibatol A, salidroside, donepezil, rivastigmine, galantamine, huperzine A, curcumin, rapamycin, memantine hydrochloride, metformin, retinoic acid, quercetin, clioquinol, simvastatin, resveratrol, tea polyphenols, ibuprofen, berberine, dasatinib, mefloquine, masitinib, and at least one of nucleic acid drugs; drugs for treating traumatic brain injury include: aspirin, simvastatin, atorvastatin, warfarin, mannitol, methylcobalamin, trimethoprim-sulfamethoxazole, neurotrophic factors At least one of the following: drugs for treating epilepsy: including at least one of phenytoin sodium, carbamazepine, phenobarbital, sodium valproate, ethosuximide, clonazepam, oxcarbazepine, lamotrigine, levetiracetam, topiramate, felbamate, galapentin, and gabapentin; drugs for treating Huntington's disease: including at least one of tetrabenazine, tritium tetrabenazine, and nucleic acid drugs; drugs for treating amyotrophic lateral sclerosis: including riluzole, eladarone, baclofen, diazepam, trihexyphenidyl, amitriptyline, and benzodiazepine. At least one of sodium butyrate and taurine diol; drugs for treating Parkinson's disease: including at least one of antan, amantadine, madopar, clozapine, quetiapine, ziprasidone, rivastigmine, donepezil, pimaseline, aripiprazole, and piribedil; drugs for treating multiple sclerosis: including at least one of dexamethasone, methylprednisolone, cyclophosphamide, and prednisone; drugs for treating depression: including at least one of paroxetine, sertraline, fluoxetine, and fluvoxamine; drugs for treating neurosis Drugs for treating inflammation: including at least one of methylcobalamin, adenosylcobalamin, vitamin B12, and aspirin; drugs for treating cerebrovascular diseases: including at least one of lovastatin, aspirin, ropyrazole, mannitol, and piracetam; drugs for treating hydrocephalus: including at least one of acetazolamide, mannitol, and glycerol fructose; drugs for treating brain tumors: including at least one of paclitaxel, doxorubicin, temozolomide, methotrexate, fluorouracil, cytarabine, ibrutinib, and hydroxychloroquine.

[0017] Furthermore, the surface of the drug-loaded nanoparticles is also bound to a brain-targeting polypeptide; optionally, the brain-targeting polypeptide is selected from Angiopep-2 peptide and / or RVG29 peptide.

[0018] The present invention also provides a method for preparing any of the above-mentioned NK cell drug delivery systems, comprising mixing the activated NK cells and the drug-loaded nanoparticles, and incubating them to prepare the NK cell drug delivery system.

[0019] Furthermore, the preparation method satisfies one or more of the following (1)-(2):

[0020] (1) Every 1×10 5 -2×10 6 NK cells are mixed with more than 0 mg and less than or equal to 0.2 mg of drug-loaded nanoparticles. Preferably, every 2×10 6 0.03mg-0.2mg of drug-loaded nanoparticles were mixed into each NK cell;

[0021] (2) The incubation temperature is 30-40°C and the time is 10-45 minutes.

[0022] Furthermore, every 2×10 6 0.125 mg of drug-loaded nanoparticles were mixed into each NK cell.

[0023] Furthermore, the drug-loaded nanoparticles are prepared by self-assembly of a polymer carrier or albumin. Preferably, the preparation method of the drug-loaded nanoparticles comprises dissolving the drug, the first polymer material, and phospholipid-thioketal-polyethylene glycol-maleimide in an organic solvent, adding water dropwise under ultrasonic conditions, and dialyzing to remove the organic solvent and free drug to obtain a suspension containing the drug-loaded nanoparticles. More preferably, the preparation method of the drug-loaded nanoparticles further satisfies at least one of the following A and F conditions:

[0024] A. Ultrasonic power is 10-30W, preferably 30W;

[0025] B. Dialysis time is 24-72h, preferably 48h;

[0026] C, the organic solvent is selected from one or more of ethanol and dimethyl sulfoxide, preferably a mixed solvent of ethanol and dimethyl sulfoxide in a volume ratio of 1:8-10;

[0027] D. The mass ratio of the drug, the first polymer material, and the phospholipid-thioketal-polyethylene glycol-maleimide is 3-7:3-15:3-15, preferably 5:10:10;

[0028] E. The ratio of the mass of the drug to the volume of the organic solvent and water is 3-7 mg: 0.5-2.5 mL: 2-10 mL;

[0029] F. After dialysis, the method further comprises mixing the brain-targeting polypeptide with a suspension containing drug-loaded nanoparticles, and ultrafiltration to remove free brain-targeting polypeptide in the drug-loaded nanoparticles to obtain targeted drug-loaded nanoparticles; preferably, the mixing time is 0.5-4 hours.

[0030] Furthermore, the ultrasonication time is more than 30 seconds, for example, 30 seconds to 2 minutes.

[0031] The preparation method of the activated NK cells comprises mixing the NK cells with a solution containing a thiol reducing agent and incubating the mixture; preferably, the incubation temperature is 30-37° C. and the time is 10-45 min; preferably, the amount of activated NK cells per 1×10 6 -10×10 6 0.3-4 mg of a sulfhydryl reducing agent is mixed into each cell; preferably, the solution containing the sulfhydryl reducing agent is a PBS solution containing the sulfhydryl reducing agent; preferably, the concentration of the sulfhydryl reducing agent in the solution containing the sulfhydryl reducing agent is 0.1-0.5 mg / mL.

[0032] The present invention also provides the use of any of the above-mentioned NK cell drug delivery systems or the NK cell drug delivery systems prepared by any of the above-mentioned preparation methods in preparing diagnostic reagents for brain diseases or preparing drugs for preventing or treating brain diseases. Beneficial effects

[0033] 1. The NK cell drug delivery system provided by the present invention comprises activated NK cells and drug-loaded nanoparticles bound to the surface of the activated NK cells, wherein the activated NK cells are NK cells containing thiol groups on their surface, and the drug-loaded nanoparticles contain maleimide groups or thiol groups on their surface. The drug-loaded nanoparticles bind to the thiol groups on the surface of the activated NK cells through the maleimide groups or thiol groups, significantly improving the ability of the drug-loaded nanoparticles to cross the blood-brain barrier and enter the brain inflammatory lesions through the NK cells; in addition, NK cells can achieve specific cell therapy by recognizing target cells in inflammatory lesions, such as infected cells or inflammatory cells; more importantly, NK cells can secrete a variety of cytokines and mediators, regulate immune responses and reduce inflammatory responses, thereby affecting the development of diseases and therapeutic effects at multiple levels, thereby producing a stronger therapeutic effect. The drug-loaded nanoparticles are chemically coupled to the surface of NK cells and cross the blood-brain barrier by "hitchhiking" to reach the inflammatory sites in the brain, thereby improving drug utilization and reducing the toxic side effects of free drugs on the body. In summary, the NK cell drug delivery system of the present invention has high targeting and low biotoxicity to brain disease cells or tissues through NK cells and drug-loaded nanoparticles that combine maleimide groups or thiol groups with thiol groups, significantly improving the effect of treating brain diseases.

[0034] 2. The NK cell drug delivery system provided by the present invention optimizes the raw materials of the polymer carrier, namely, includes a first polymer material and phospholipid-ketalthioglycolate-polyethylene glycol-maleimide, wherein the first polymer material is selected from at least one of polylactic acid-glycolic acid copolymer, polylactic acid, and polycaprolactone; in particular, distearoylphosphatidylethanolamine-ketalthioglycolate-polyethylene glycol-maleimide and polylactic acid-glycolic acid copolymer (PLGA50k, lactic acid:glycolic acid = 50:50) are used as raw materials, and when the mass ratio of the first polymer material to phospholipid-ketalthioglycolate-polyethylene glycol-maleimide is controlled to be 3-15:3-15 (especially 1:1), the drug can be efficiently loaded into the polymer particles, forming stable, high-encapsulation-efficiency, high-loaded drug-loaded nanoparticles, and the drug-loaded nanoparticles have good biocompatibility and biodegradability; in addition, the drug-loaded nanoparticles can be shed and released from the surface of NK cells under the action of oxidative stress (ROS) at the inflammatory site, thereby better exerting their effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0036] Figure 1 is a particle size distribution diagram of drug-loaded nanoparticles RVG-NPs prepared in Example 1. The abscissa "Size" represents the particle size, and the ordinate "Intensity" represents the content percentage.

[0037] Figure 2 is a confocal fluorescence imaging of drug-loaded nanoparticles RVG-NPs connected to NK cells with and without TCEP treatment in Experimental Example 3.

[0038] Figure 3 shows the statistical graph and fluorescence imaging of the efficiency of NK cell binding of RVG-NPs at different concentrations in Experimental Example 2. The horizontal axis is RVG-NPs concentration, and the vertical axis is fluorescence intensity.

[0039] Figure 4 shows the optical microscope (A) and fluorescence microscope (B) images of RVG-NPs and RVG-NPs@NK cell treated groups in Experimental Example 4, which were detected by Transwell experiment to pass through the simulated BBB cell layer in vitro and reach the lower layer of Transwell culture medium and lower layer cells.

[0040] Figure 5 is a statistical graph showing the number of NK cells in the RVG-NPs and RVG-NPs@NKcell treated groups that passed through the simulated BBB cell layer in vitro and reached the lower layer of the Transwell culture medium in Experimental Example 4 detected by the Transwell experiment.

[0041] Figure 6 shows the fluorescence statistics of RVG-NPs and RVG-NPs@NK cells in the Transwell assay, as measured in Experimental Example 4, after the RVG-NPs and RVG-NPs@NK cells-treated groups passed through the simulated BBB cell layer in vitro and reached the lower layer of the Transwell. α-syn (+) represents the group treated with α-syn protein, while α-syn (-) represents the group not treated with α-syn protein.

[0042] Figure 7 shows brain fluorescence images (A) and fluorescence intensity statistics (B) of wild-type mice and Parkinson's disease (PD) model mice 9 hours after tail vein injection of RVG-NPs@NK cells in Experimental Example 5. Control mice represent wild-type mice; PD mice represent PD model mice. The vertical axis (FL intensity) represents fluorescence intensity.

[0043] Figure 8 shows brain fluorescence imaging at different time points after tail vein injection of RVG-NPs and RVG-NPs@NK cells in PD model mice in Experimental Example 6.

[0044] Figure 9 shows the statistical graph of brain fluorescence intensity at different time points after tail vein injection of RVG-NPs and RVG-NPs@NK cells in PD model mice in Experimental Example 6. The vertical axis, Relative Fluorescence intensity, is the relative fluorescence intensity.

[0045] Figure 10 is the fluorescence imaging of the main organs of PD model mice 24 hours after tail vein injection of RVG-NPs and RVG-NPs@NK cells in Experimental Example 6.

[0046] Figure 11 is a statistical graph of the fluorescence intensity in major organs of PD model mice 24 hours after tail vein injection of RVG-NPs and RVG-NPs@NK cells in Experimental Example 6. The vertical axis, Relative Fluorescence intensity, is the relative fluorescence intensity.

[0047] In the above figures, Heart, Liver, Spleen, Lung, Kidney, and Brain represent heart, liver, spleen, lung, kidney, and brain respectively.

[0048] * indicates a significant difference between the two groups (p<0.05), ** indicates p<0.01, and *** indicates p<0.001. Modes for Carrying Out the Invention

[0049] The technical solutions of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. It is apparent that the embodiments described are only some, not all, of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. In the present invention, the term ICG stands for indocyanine green.

[0050] Example 1 Preparation of drug-loaded nanoparticles

[0051] This embodiment provides a method for preparing drug-loaded nanoparticles, comprising the following steps:

[0052] (1) The simulated drug ICG (5 mg), poly(lactic acid-glycolic acid) copolymer (PLGA50k, lactic acid:glycolic acid = 50:50, 10 mg), and ROS-responsive phospholipid (distearoylphosphatidylethanolamine-thioketal-polyethylene glycol-maleimide, DSPE-TK-PEG2k-Mal, 10 mg) were weighed and dissolved in 1 mL of a mixed solvent of ethanol and dimethyl sulfoxide (1 / 9, v / v).

[0053] (2) Under ultrasonic conditions (power: 30 W, time: 1 min), ultrapure water (5 mL) was added dropwise to the above mixed solution.

[0054] (3) The mixture was dialyzed against ultrapure water (48 h) (dialysis bag MWCO = 3500 Da) to remove the organic solvent and free drug, and obtain a drug-loaded nanoparticle (NPs) suspension.

[0055] (4) RVG29 peptide (2 μg) was added to the drug-loaded nanoparticle (NPs) suspension and mixed for 2 h. The free RVG29 peptide in the drug-loaded nanoparticles was removed by ultrafiltration to obtain targeted drug-loaded nanoparticles (RVG-NPs).

[0056] The drug-loaded nanoparticles were characterized using a particle size analyzer and their particle size distribution (see Figure 1). They were then stored at 4°C until ready for use. Figure 1 shows that the RVG-NPs had a particle size distribution between 80 and 130 nm, with an average particle size of 105 nm. The polydispersity index (PDI) was 0.2 ± 0.1.

[0057] Example 2 Preparation of drug-loaded nanoparticles

[0058] This embodiment provides a method for preparing drug-loaded nanoparticles, comprising the following steps:

[0059] Albumin (HSA) was dissolved in water to prepare 5 mL of an aqueous solution with a concentration of 0.375 mg / mL to obtain an aqueous phase; then 5 mg of ICG was dissolved in 1 mL of a mixed solution of dichloromethane / ethanol (volume ratio 9:1) to obtain an oil phase; the oil phase was added dropwise to the aqueous phase while shearing for 2 minutes, and the organic solvent was removed by thin film evaporation to obtain drug-loaded nanoparticles.

[0060] Example 3 Preparation of drug-loaded nanoparticles

[0061] This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of Example 1, except that the mass of the simulated drug ICG in step (1) is adjusted from 5 mg to 2 mg.

[0062] Example 4 Preparation of drug-loaded nanoparticles

[0063] This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of Example 1, except that the mass of the ROS-responsive phospholipid in step (1) is adjusted from 10 mg to 3 mg.

[0064] Example 5 Preparation of drug-loaded nanoparticles

[0065] This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of Example 1, except that the mass of the polylactic acid-glycolic acid copolymer in step (1) is adjusted from 10 mg to 3 mg.

[0066] Example 6 Preparation of drug-loaded nanoparticles

[0067] This example provides a method for preparing drug-loaded nanoparticles, which is essentially the same as that of Example 1, except that the ultrasonic power in step (2) is adjusted from 30 W to 10 W. The particle size distribution of the prepared RVG-NPs is determined to be between 165 and 205 nm.

[0068] Example 7 Preparation of drug-loaded nanoparticles

[0069] This example provides a method for preparing drug-loaded nanoparticles, which is essentially the same as that of Example 1, except that the ultrasonic power in step (2) is adjusted from 30 W to 20 W. The particle size distribution of the prepared RVG-NPs is determined to be between 140 and 180 nm.

[0070] Example 8 Preparation of NK cell drug delivery system

[0071] This embodiment provides a method for preparing an NK cell drug delivery system, comprising the following steps:

[0072] (1) NK cells (2 mL, total number of cells 2×10 6The NK cells were added into PBS (5 mL) containing tris(2-carboxyethyl)-phosphine hydrochloride (TCEP, concentration: 0.3 mg / mL), incubated at 37°C for 30 min, and then washed three times with PBS. Finally, the TCEP-treated NK cells were dispersed into 0.1 mL of PBS.

[0073] (2) A PBS dispersion of RVG-NPs (prepared according to the method in Example 1) with a volume of 0.1 mL and a concentration of 1.25 mg / mL was added to the TCEP-treated NK cells described above and mixed. The mixture was incubated at 37°C for 30 min. The NK cell drug delivery system was washed three times with PBS to remove the unattached drug-loaded nanoparticles, thereby obtaining the NK cell drug delivery system (RVG-NPs@NK cell).

[0074] Experimental Example 1

[0075] The particle size, drug loading capacity, and encapsulation efficiency of the drug-loaded nanoparticles were tested. The specific testing methods for drug loading and encapsulation efficiency are as follows: After freeze-drying, the drug-loaded nanoparticles were weighed and dissolved in dimethyl sulfoxide (DMSO). The absorbance of the drug at the maximum absorption peak was measured using a UV spectrophotometer, and the drug loading capacity and encapsulation efficiency of the drug-loaded nanoparticles were calculated according to the following formulas. The drug loading capacity and encapsulation efficiency results are shown in Table 1.

[0076]

[0077] Table 1 Characterization results of drug-loaded nanoparticles in various examples

[0078] Item Particle size distribution Encapsulation efficiency Drug loading Example 1 140-180nm 90% 28% Example 2 79-100nm 87% 7% Example 3 125-175nm 88% 8% Example 4 310-450nm 78% 23% Example 5 137-189nm 67% 10%

[0079] Comparing Example 2 with Example 1 and Examples 3-5, under the same dosage, the encapsulation efficiency and drug loading of the polymer drug-loaded nanoparticles prepared using PLGA50k / DSPE-TK-PEG2k-Mal are significantly improved compared to the drug-loaded nanoparticles prepared using HSA as the raw material. By comparing Example 1 with Examples 3-5, it can be seen that the size of the polymer drug-loaded nanoparticles obtained by controlling the mass ratio of the drug ICG, polylactic acid-glycolic acid copolymer and ROS-responsive phospholipids in Example 1 is appropriate and the drug loading is the highest. By comparing Example 1 and Examples 6-7, it can be seen that the ultrasonic power has little effect on the size of the polymer drug-loaded nanoparticles, and drug-loaded nanoparticles with uniform particle size distribution can be obtained under 10W-30W.

[0080] Experimental Example 2 Fluorescence Intensity

[0081] The effects of different drug-loaded nanoparticle concentrations (or drug amounts) on the preparation of NK cell drug delivery systems were investigated. During the preparation of the NK cell drug delivery system, 1 mL of PBS solution or 1 mL of PBS dispersion of drug-loaded nanoparticles RVG-NPs at concentrations of 31.25, 62.5, 125, or 162.5 μg / mL was added. The remaining processes and conditions were the same as those in Example 8. The NK cell drug delivery system (RVG-NPs@NK cell) constructed in Example 8 was added to a 24-well plate, and the fluorescence intensity was detected using small animal imaging to calculate the loading efficiency (as shown in Figure 3).

[0082] Figure 3 shows the statistical results of the NK cell drug loading efficiency prepared in Example 8. From Figure 3 we can see that its maximum loading efficiency is 62.5 μg / 10 6 cells, the fluorescence intensity was about 1.0×10 7 .

[0083] Experimental Example 3 Morphological Analysis

[0084] 1. Experimental methods

[0085] NK cells were randomly divided into two groups, TCEP+ group and TCEP- group. In the TCEP+ group, NK cells were treated with TCEP according to the method of Example 8 and then mixed with drug-loaded nanoparticles RVG-NPs to obtain an NK cell drug delivery system. In the TCEP- group, NK cells were not treated with TCEP and directly mixed with drug-loaded nanoparticles RVG-NPs to obtain the experimental results. The only difference from Example 8 was that step (1) was omitted. Then, 4% paraformaldehyde was added to the above NK cells for fixation, and DAPI was added to stain the NK cell nuclei. The morphological changes of the two groups of NK cells and the efficiency of grafting drug-loaded nanoparticles on the NK cell surface were observed using a confocal microscope (as shown in Figure 2).

[0086] 2. Experimental results

[0087] Figure 2 shows the observation results of confocal microscopy in Example 8. The blue fluorescence originates from the NK cell nucleus, and the red punctate fluorescence originates from RVG-NPs. As can be seen from Figure 2, in the experimental group of NK cells treated with TCEP, the punctate fluorescence of RVG-NPs is basically distributed on the surface of the NK cell membrane (indicated by the white arrows), proving that the TCEP treatment method can effectively graft RVG-NPs on the surface of NK cells; while in the control group of NK cells not treated with TCEP, most of the RVG-NPs punctate fluorescence is distributed in the cytoplasm, indicating that the RVG-NPs are taken up by NK cells and enter the cytoplasm. The above experimental results demonstrate the successful construction of the NK cell drug delivery system.

[0088] Experimental Example 4 NK in vitro migration assay

[0089] 1. Experimental methods

[0090] To establish an in vitro BBB and simulate PD lesion models: First, 100,000 bEnd.3 cells (mouse brain microvascular endothelial cells) were plated in a Transwell chamber and cultured until the cells adhered tightly. Then, 100,000 BV2 cells (mouse microglia) and 100,000 MN9D cells (mouse midbrain dopaminergic neurons) were plated in a 24-well plate below the Transwell chamber. After the cells adhered, α-synuclein (α-synuclein, a pathological hallmark of Parkinson's disease (PD) and its accumulation in the brain) was added to the culture medium below the Transwell chamber. The Transwell chamber was then placed in the lower 24-well plate and cultured for 24 hours. Then, 30 μL of the NK cell drug delivery system (RVG-NPs@NK cell) prepared in Example 8 or 30 μL of a 1.25 mg / mL dispersion of drug-loaded RVG-NPs (prepared by the method in Example 1) in PBS was added to the Transwell chamber. A control, treated with RVG-NPs@NK cells but without α-syn protein, was treated under the same conditions. After 6 hours, the supernatant and cells in the lower layer were separated. Polylysine was added to the supernatant and cells in the lower layer to fix the cells. One hour later, the number of NK cells that had crossed the BBB in the supernatant was observed and counted using an optical microscope. The nuclei of the cells in the lower layer were stained with DAPI, and the fluorescence intensity of the RVG-NPs in the cells was then observed and counted using a fluorescence microscope.

[0091] 2. Experimental results

[0092] As shown in Figures 4A and 5, the NK cell crossing efficiency was significantly improved in the presence of α-syn, demonstrating the NK cell drug delivery system's ability to home to PD lesions or inflammatory sites. As shown in Figures 4B and 6, the red fluorescence signal (derived from RVG-NPs) in the cytoplasm of the lower layer cells (BV2 cells and MN9D cells) treated with the NK cell drug delivery system increased significantly, indicating that the NK cell drug delivery system can effectively carry drug-loaded nanoparticles across the BBB in vitro. Furthermore, after entering the PD lesions, the drug-loaded nanoparticles can be shed from the NK cell surface and ultimately taken up by the target cells (BV2 cells and MN9D cells).

[0093] Experimental Example 5: NK targeting brain lesions in vivo

[0094] 1. Experimental methods

[0095] Six C57 mice were randomly divided into an experimental control group (Control) and a Parkinson's disease model group (PD mice group or PD model group) according to sex and weight, with 3 mice in each group. For the mice in the PD model group, AAV-A53T α-Syn virus (3 TCID50, 0.5 μL) was injected through brain localization; for the mice in the experimental control group, control virus (AAV, 0.5 μL) was injected through brain localization, and penicillin was injected into the muscle. One month after the virus injection, the mouse Parkinson's disease model was successfully established. CM-DiI-labeled NK cells (injection dose of 2×10 6 The mouse brains were removed 9 hours later for fluorescence imaging and fluorescence intensity analysis.

[0096] 2. Experimental results

[0097] The results are shown in Figure 7. As can be seen from the fluorescence imaging and fluorescence intensity statistics in Figure 7, the fluorescence intensity of the brain tissue of the PD model group was higher than that of the experimental control group, indicating that more NK cells were enriched in the brain tissue of the PD model mice or accumulated in the brain lesions with inflammation.

[0098] Experimental Example 6: In vivo targeting of brain lesions by NK cell drug delivery system

[0099] 1. Experimental methods

[0100] Parkinson's disease (PD) mouse models were established according to the method of Experimental Example 5. The PD model mice were randomly divided into a drug-loaded nanoparticle (RVG-NPs) group and a NK cell drug delivery system (RVG-NPs@NK cell) group. The RVG-NPs group received a suspension of RVG-NPs (drug-loaded nanoparticle concentration of 1.25 mg / mL) via tail vein injection; the RVG-NPs@NK cell group received RVG-NPs@NK cells prepared in Example 8 via tail vein injection, with a dosing volume of 150 μL in both groups. Fluorescence intensity of brain tissue was observed and measured using a small animal imaging device at 1, 3, 6, 9, 12, and 24 hours. Major organs were removed at 24 hours for fluorescence imaging and fluorescence intensity analysis.

[0101] 2. Experimental results

[0102] From the results in Figures 8-9, it can be seen that the brain accumulation efficiency of the NK cell drug delivery system is significantly higher than that of the drug-loaded nanoparticle group, proving that the NK cell drug delivery system can efficiently accumulate in the inflammatory site in the brain.

[0103] From the results in Figures 10-11, it can be seen that there is no significant difference in the distribution of NK cell drug delivery system and drug-loaded nanoparticles in the main organs, but the fluorescence intensity of NK cell drug delivery system in the brain is 3.3×10 8 The fluorescence intensity of drug-loaded nanoparticles in the brain is 1.1×10 8 The accumulation efficiency of the NK cell drug delivery system in the brain is about three times that of drug-loaded nanoparticles, which proves that the use of NK cell drug delivery strategy can significantly improve the accumulation efficiency of drugs in the brain, and is expected to improve the treatment effect of brain diseases.

[0104] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A NK cell drug delivery system, characterized in that: It includes activated NK cells and drug-loaded nanoparticles bound to the surface of the activated NK cells. The activated NK cells are NK cells containing thiol groups on the surface. The surface of the drug-loaded nanoparticles contains maleimide groups or thiol groups. The drug-loaded nanoparticles are bound to the thiol groups on the surface of the activated NK cells through the maleimide groups or thiol groups.

2. The NK cell drug delivery system according to claim 1, characterized in that The activated NK cells are prepared by reducing the disulfide bonds of NK cell surface proteins to sulfhydryl groups using a sulfhydryl reducing agent; optionally, the sulfhydryl reducing agent is selected from one or more of tris(2-carboxyethyl)-phosphine hydrochloride, mercaptoethanol, dithiothreitol, dimercaptopropanol, β-mercaptoethylamine and cysteine.

3. The NK cell drug delivery system according to claim 1, characterized in that The drug-loaded nanoparticles include a carrier shell and a drug contained in the carrier shell, wherein the carrier shell includes a polymer carrier or albumin, wherein the polymer carrier includes a first polymer material and phospholipid-ketalthioethanol-polyethylene glycol-maleimide, and the first polymer material is selected from at least one of polylactic acid-co-glycolic acid, polylactic acid, and polycaprolactone; Preferably, the mass ratio of the first polymer material to phospholipid-thioketal-polyethylene glycol-maleimide is 3-15:3-15; Preferably, the phospholipid-thioketal-polyethylene glycol-maleimide is distearoylphosphatidylethanolamine-thioketal-polyethylene glycol-maleimide; Preferably, the mass ratio of the drug to the polymer carrier or albumin is 3-7:2-30.

4. The NK cell drug delivery system according to claim 1, characterized in that The drug in the drug-loaded nanoparticles includes one or more of simulated drugs and drugs for preventing or treating brain diseases.

5. The NK cell drug delivery system according to any one of claims 1 to 4, characterized in that: The surface of the drug-loaded nanoparticles is also bound to a brain-targeting polypeptide; optionally, the brain-targeting polypeptide is selected from Angiopep-2 peptide and / or RVG29 peptide.

6. The method for preparing the NK cell drug delivery system according to any one of claims 1 to 5, characterized in that: The method comprises mixing the activated NK cells and the drug-loaded nanoparticles, and incubating them to prepare the NK cell drug delivery system.

7. The method for preparing the NK cell drug delivery system according to claim 6, characterized in that: The preparation method satisfies one or more of the following (1)-(2): (1) Every 1×10 5 -2×10 6 NK cells are mixed with more than 0 mg and less than or equal to 0.2 mg of drug-loaded nanoparticles. Preferably, each 2×10 6 0.03mg-0.2mg of drug-loaded nanoparticles were mixed into each NK cell; (2) The incubation temperature is 30-40°C and the time is 10-45 minutes.

8. The method for preparing the NK cell drug delivery system according to claim 6, characterized in that: The drug-loaded nanoparticles are prepared by self-assembly of a polymer carrier or albumin. Preferably, the preparation method of the drug-loaded nanoparticles comprises dissolving the drug, the first polymer material and phospholipid-ketalthioethanol-polyethylene glycol-maleimide in an organic solvent, dripping water under ultrasonic conditions, and dialyzing to remove the organic solvent and free drugs to obtain a suspension containing the drug-loaded nanoparticles; more preferably, the preparation method of the drug-loaded nanoparticles further satisfies at least one of the following A to F: A. Ultrasonic power is 10-30W, preferably 30W; B. Dialysis time is 24-72h, preferably 48h; C. The organic solvent is selected from one or more of ethanol and dimethyl sulfoxide, preferably a mixed solvent of ethanol and dimethyl sulfoxide in a volume ratio of 1:8-10; D. The mass ratio of the drug, the first polymer material and the phospholipid-thioketal-polyethylene glycol-maleimide is 3-7:3-15:3-15, preferably 5:10:10; E. The ratio of the mass of the drug to the volume of the organic solvent and water is 3-7 mg: 0.5-2.5 mL: 2-10 mL; F. After dialysis, the method further includes mixing the brain-targeting polypeptide with a suspension containing drug-loaded nanoparticles, and removing free brain-targeting polypeptide in the drug-loaded nanoparticles by ultrafiltration to obtain targeted drug-loaded nanoparticles; preferably, the mixing time is 0.5-4h.

9. The method for preparing the NK cell drug delivery system according to claim 7, characterized in that: The preparation method of the activated NK cells comprises mixing the NK cells with a solution containing a thiol reducing agent, and incubating the mixture; preferably, the incubation temperature is 30-37° C., and the time is 10-45 min; preferably, each 1×10 6 -10×10 6 0.3-4 mg of thiol reducing agent is mixed into each cell; preferably, the solution containing the thiol reducing agent is a PBS solution containing the thiol reducing agent; preferably, the concentration of the thiol reducing agent in the solution containing the thiol reducing agent is 0.1-0.5 mg / mL.

10. Use of the NK cell drug delivery system described in any one of claims 1 to 5 or the NK cell drug delivery system prepared by the preparation method described in any one of claims 6 to 9 in preparing diagnostic reagents for brain diseases or preparing drugs for preventing or treating brain diseases.

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