本发明属于药物领域，具体涉及一种NK细胞药物递送系统及其制备方法和用途。The present invention belongs to the field of medicines, and in particular relates to a NK cell drug delivery system and a preparation method and use thereof.
目前脑部疾病全球发病率高且不断上升，市场庞大。但是脑部疾病药物开发仍然具有挑战性，其开发成本高、临床使用路径长、失败率高。脑部受到生理屏障（特别是血脑屏障）的高度保护，而脑部疾病药物的研发难点主要在于血脑屏障。98%以上小分子药物难以通过血脑屏障进入中枢系统。没有主动运输，药物无法进入脑内，导致大量体外有效的药物无法通过血脑屏障而被提前宣告无效。更重要的是，药物穿越血脑屏障后还需要进一步靶向到病灶细胞。如何将药物更好地聚集在病灶细胞而不损伤正常的脑细胞，是另一个难点。如何帮助药物突破血脑屏障，提高脑部病灶部位的靶向效率，已成为脑部疾病治疗的关键。At present, the global incidence of brain diseases is high and rising, and the market is huge. However, the development of brain disease drugs is still challenging, with high development costs, long clinical use paths, and high failure rates. The brain is highly protected by physiological barriers (especially the blood-brain barrier), and the difficulty in the development of brain disease drugs lies mainly in the blood-brain barrier. More than 98% of small molecule drugs have difficulty passing through the blood-brain barrier 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 unable to pass through the blood-brain barrier and being declared invalid in advance. More importantly, after crossing the blood-brain barrier, the drug needs to be further targeted to the lesion cells. How to better aggregate the drug in the lesion cells without damaging normal brain cells is another difficulty. How to help drugs break through the blood-brain barrier and improve the targeting efficiency of brain lesions has become the key to the treatment of brain diseases.
针对目前脑部疾病的治疗主要有两种方式：手术以及小分子药物系统性或局部给药。对于一些脑部炎症疾病，如脑脓肿、脑肿瘤等，手术治疗可以通过切除病灶或引流脓液来达到治疗目的。但是手术治疗具有一定的风险，可能会导致手术相关的并发症和副作用。此外，部分脑部疾病不适合手术治疗，如脑膜炎、脑炎以及神经退行性疾病等。小分子药物治疗是最常见和常规的治疗方法之一。但是多数药物都难以有效地穿越血脑屏障，降低了药物的治疗效果；此外，药物无法直接修复受损的脑部组织，仅能减轻炎症症状；一些药物可能存在副作用和毒性反应；部分病原体可能产生耐药性，导致治疗效果下降。There are currently two main ways to treat 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 treatment goal by removing the lesions 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 are difficult to effectively cross the blood-brain barrier, which reduces the therapeutic effect of the drugs; in addition, drugs cannot directly repair damaged brain tissue, but can only alleviate inflammatory symptoms; some drugs may have side effects and toxic reactions; some pathogens may develop drug resistance, resulting in reduced treatment effects.
近年来，细胞递药系统的快速发展为脑部疾病的治疗提供了新思路。尽管血脑屏障 (BBB) 和血脑脊髓液屏障 (BCSFB) 严格限制细胞和分子进入脑实质，但免疫细胞可以穿过这些屏障，尤其是在中风和炎症等病理条件下。另外，细胞递药系统作为一种新型的治疗策略，具有许多优势。1) 靶向性：细胞递药系统可以被设计成具有针对性的治疗效应，通过识别和靶向疾病细胞或组织，提供更准确和有效的治疗。2) 个体化治疗：由于细胞载体通常来源于患者自身的细胞或经过个体化处理的外源性细胞，因此可以实现个体化的治疗策略，考虑到患者的独特生物学特征和需求。3) 克服免疫逃逸：细胞递药系统能够克服一些传统药物面临的免疫逃逸问题。例如，肿瘤细胞可能通过改变表面抗原表达或其他机制来逃避免疫攻击，而细胞药物可以通过多种机制来识别和攻击这些逃逸的细胞。4) 长效效应：细胞药物可以在体内长时间存活和活动，提供持久的治疗效果。相比之下，一些传统药物可能需要频繁的剂量给药才能维持治疗效果。5) 多种治疗机制：细胞药物可以通过多种机制发挥治疗作用，如直接杀伤疾病细胞、分泌细胞因子和细胞间信号分子、调节免疫反应等，从而在多个层面上影响疾病的发展和治疗效果。如何设计和构建新型针对脑部疾病的细胞递药系统，提高治疗脑部疾病的效果，是本发明要解决的技术问题和亟需实现的目标。In recent years, the rapid development of cell-based drug delivery systems has provided new ideas 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, especially in pathological conditions such as stroke and inflammation. In addition, as a new therapeutic strategy, cell-based drug delivery systems have many advantages. 1) Targetedness: Cell-based drug delivery systems can be designed to have targeted therapeutic effects, providing more accurate and effective treatments by identifying and targeting disease cells or tissues. 2) Personalized treatment: Since cell carriers are usually derived from the patient's own cells or exogenous cells that have been personalized, personalized treatment strategies can be achieved, 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 problems faced by some traditional drugs. For example, tumor cells may escape immune attacks by changing surface antigen expression or other mechanisms, while cell-based drugs can recognize and attack these escaped cells through a variety of mechanisms. 4) Long-lasting effect: Cell-based drugs can survive and act in the body for a long time, providing lasting therapeutic effects. In contrast, some traditional drugs may require frequent dosing to maintain therapeutic effects. 5) Multiple therapeutic mechanisms: Cellular drugs can exert therapeutic effects through multiple mechanisms, such as directly killing disease cells, secreting cytokines and intercellular signaling molecules, regulating immune responses, etc., thereby affecting the development of diseases and therapeutic effects at multiple levels. How to design and construct a new cell drug delivery system for brain diseases and improve the effect of treating brain diseases is a technical problem to be solved by the present invention and a goal to be achieved urgently.
为了克服现有药物对于脑部疾病治疗效果较差的技术问题，本发明提出了一种新型的NK细胞药物递送系统及其制备方法，该NK细胞药物递送系统对脑部疾病细胞或者组织具有高度的靶向性、低生物毒性，制备工艺简单，显著提高治疗脑部疾病的效果。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 a preparation method thereof. The NK cell drug delivery system has high targeting and low biological toxicity to brain disease cells or tissues, a simple preparation process, and significantly improves the effect of treating brain diseases.
本发明提供了一种NK细胞药物递送系统，其包括活化的NK细胞和结合在所述活化的NK细胞表面的载药纳米颗粒，所述活化的NK细胞为表面含巯基的NK细胞，所述载药纳米颗粒表面含有马来酰亚胺基团或者巯基基团，所述载药纳米颗粒通过马来酰亚胺基团或者巯基基团与活化的NK细胞表面的巯基相结合。The present invention provides a NK cell drug delivery system, which comprises activated NK cells and drug-loaded nanoparticles bound to the surfaces of the activated NK cells, wherein the activated NK cells are NK cells containing thiol groups on the surfaces, the drug-loaded nanoparticles contain maleimide groups or thiol groups on the surfaces of the activated NK cells through the maleimide groups or thiol groups.
进一步地，所述活化的NK细胞是通过巯基类还原剂将NK细胞表面蛋白的二硫键还原为巯基而制成；可选的，三（2-羧乙基）-膦盐酸盐、巯基乙醇、二硫苏糖醇、二巯基丙醇、β巯基乙胺和半胱氨酸中的一种或者多种。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.
进一步地，所述载药纳米颗粒包括载体外壳和包载于载体外壳内的药物，所述载体外壳包含聚合物载体或者白蛋白，其中，所述聚合物载体包括第一聚合物材料以及磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺，所述第一聚合物材料选自聚乳酸-羟基乙酸共聚物（PLGA）、聚乳酸（PLA）、聚己内酯（PCL）中的至少一种；优选的，所述第一聚合物材料与磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺的质量比为3-15:3-15；优选的，所述磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺为二硬脂酰基磷脂酰乙醇胺-酮缩硫醇-聚乙二醇-马来酰亚胺；优选的，所述药物的质量与聚合物载体或者白蛋白的质量比为3-7：2-30。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-ketethiolate-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-ketethiolate-polyethylene glycol-maleimide is 3-15:3-15; preferably, the phospholipid-ketethiolate-polyethylene glycol-maleimide is distearoylphosphatidylethanolamine-ketethiolate-polyethylene glycol-maleimide; preferably, the mass ratio of the drug to the polymer carrier or albumin is 3-7:2-30.
更优选的，所述第一聚合物材料与磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺的质量比为1:1。More preferably, the mass ratio of the first polymer material to phospholipid-thioketal-polyethylene glycol-maleimide is 1:1.
优选的，所述药物、第一聚合物材料与磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺的质量比为3-7：3-15：3-15，更优选为5：10：10。Preferably, the mass ratio of the drug, the first polymer material and phospholipid-thioketal-polyethylene glycol-maleimide is 3-7:3-15:3-15, more preferably 5:10:10.
可选的，所述第一聚合物材料的分子量为20k-100k。Optionally, the molecular weight of the first polymer material is 20k-100k.
具体的，第一聚合物材料可以是PLGA50k、PLGA20k、PLGA100k、PLA50k、PCL50k等。Specifically, the first polymer material may be PLGA50k, PLGA20k, PLGA100k, PLA50k, PCL50k, etc.
进一步地，所述载药纳米颗粒中的药物包括模拟药物、预防或治疗脑部疾病的药物中的一种或者几种。Furthermore, the drug in the drug-loaded nanoparticles includes one or more of simulated drugs and drugs for preventing or treating brain diseases.
其中模拟药物，包括但不局限于荧光染料，例如芘、苝、BODIPY、荧光素、罗丹明、花菁染料、ICG系列染料及衍生物。The simulated drugs include but are not limited to fluorescent dyes, such as pyrene, perylene, BODIPY, fluorescein, rhodamine, cyanine dyes, ICG series dyes and derivatives.
所述脑部疾病包括但不局限于阿尔茨海默症，创伤性脑损伤，癫痫，亨廷顿病，肌萎缩侧索硬化，帕金森病，多发硬化症，抑郁症，神经炎症，脑血管疾病，脑积水，脑肿瘤；其中脑肿瘤包括原发性脑胶质瘤、继发性肿瘤脑转移、少突神经胶质瘤、脑膜瘤、星形细胞瘤、脑膜瘤及其他脑肿瘤。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.
其中，治疗阿尔茨海默症的药物：包括芬戈莫德、3‑正丁基苯酞及其衍生物、姜黄素、西地那非、艾塞那肽（Exendin‑4）、雷公藤红素、奥米茄-3（Omega‑3）多元不饱和脂肪酸、Malibatol A、红景天苷、多奈哌齐、卡巴拉汀、加兰他敏、石杉碱甲、姜黄素、雷帕霉素、盐酸美金刚、二甲双胍、维甲酸、懈皮素、氯碘羟喹、辛伐他丁、白藜芦醇、茶多酚、布洛芬、小檗碱、达沙替尼、甲氟喹、马赛替尼、核酸类药物中的至少一种；治疗创伤性脑损伤的药物：包括阿司匹林、辛伐他汀、托伐他汀、华法林、甘露醇、甲钴胺、三甲氧苄嗪、神经营养因子中的至少一种；治疗癫痫的药物：包括苯妥英钠、卡马西平、苯巴比妥、丙戊酸钠、乙琥胺、氯硝西泮、奥卡西平、拉莫三嗪、左乙拉西坦、托吡酯、非氨酯、加拉喷丁、加巴喷中的至少一种；治疗亨廷顿病的药物：包括丁苯喹嗪、氚丁苯那嗪、核酸类药物中的至少一种；治疗肌萎缩侧索硬化的药物：包括利鲁唑、依拉达奉、巴氯芬、地西泮、苯海索、阿米替林、苯丁酸钠、牛磺酸二醇中的至少一种；治疗帕金森病的药物：包括安坦、金刚烷胺、美多芭、氯氮平、喹硫平、齐拉西酮、卡巴拉汀、多奈哌齐、哌马色林、阿立哌唑、吡贝地尔中的至少一种；治疗多发硬化症的药物：包括地塞米松、甲泼尼龙、环磷酰胺、强的松中的至少一种；治疗抑郁症的药物：包括帕罗西汀、舍曲林、氟西汀、氟伏沙明中的至少一种；治疗神经炎症的药物：包括甲钴胺、腺苷钴胺、维生素B12、阿司匹林中的至少一种；治疗脑血管疾病的药物：包括洛伐他汀、阿司匹林、罗吡唑、甘露醇、吡拉西坦中的至少一种；治疗脑积水的药物：包括乙酰唑胺、甘露醇、甘油果糖中的至少一种；治疗脑肿瘤的药物：包括紫杉醇、阿霉素、替莫唑胺、甲氨蝶呤、氟尿嘧啶、阿糖胞苷、伊布替尼、羟氯喹中的至少一种。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; drugs for treating epilepsy: including at least one of phenytoin sodium, carbamazepine, phenobarbital, sodium valproate, ethosuximide, clonazepam, oxcarbazepine, lamotrigine, levetiracetam, topiramate, felbamate, galapentine, 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 benzathine. At least one of sodium butyrate and taurine diol; drugs for the treatment of Parkinson's disease: including at least one of antan, amantadine, madopar, clozapine, quetiapine, ziprasidone, rivastigmine, donepezil, pimaseline, aripiprazole, and piribedil; drugs for the treatment of multiple sclerosis: including at least one of dexamethasone, methylprednisolone, cyclophosphamide, and prednisone; drugs for the treatment of depression: including at least one of paroxetine, sertraline, fluoxetine, and fluvoxamine; drugs for the treatment of neurosis Drugs for 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.
进一步地，所述载药纳米颗粒的表面还结合有脑靶向多肽；可选的，所述脑靶向多肽选自Angiopep-2肽和/或RVG29肽。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.
本发明还提供了上述任一所述的NK细胞药物递送系统的制备方法，包括，将所述活化的NK细胞和所述载药纳米颗粒混合，孵育，制得NK细胞药物递送系统。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.
进一步地，所述制备方法满足如下（1）-（2）中的一项或者多项：Furthermore, the preparation method satisfies one or more of the following (1)-(2):
（1）每1×105-2×106个NK细胞中混入大于0mg小于等于0.2mg的载药纳米颗粒，优选的，每2×106个NK细胞中混入0.03mg-0.2mg的载药纳米颗粒；(1) Mixing greater than 0 mg and less than or equal to 0.2 mg of drug-loaded nanoparticles into every 1×105 -2×106 NK cells, preferably, mixing 0.03 mg-0.2 mg of drug-loaded nanoparticles into every 2×106 NK cells;
（2）孵育的温度为30-40℃，时间为10-45min。(2) The incubation temperature is 30-40°C and the time is 10-45 minutes.
进一步地，每2×106个NK细胞中混入0.125mg的载药纳米颗粒。Furthermore, 0.125 mg of drug-loaded nanoparticles were mixed into every 2×106 NK cells.
进一步地，所述载药纳米颗粒通过聚合物载体或者白蛋白自组装制成，优选的，所述载药纳米颗粒的制备方法包括，将药物、第一聚合物材料以及磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺溶解在有机溶剂中，超声条件下滴加水，透析去除有机溶剂和游离药物，获得含有载药纳米颗粒的悬液；更优选的，所述载药纳米颗粒的制备方法还满足如下A-F中的至少一项：Further, 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, adding water dropwise 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-F:
A、超声功率为10-30W，优选为30W；A. Ultrasonic power is 10-30W, preferably 30W;
B、透析时间为24-72h，优选为48h；B. Dialysis time is 24-72h, preferably 48h;
C、有机溶剂选自乙醇、二甲基亚砜中的一种或者多种，优选为体积比为1:8-10的乙醇与二甲基亚砜的混合溶剂；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、所述药物、第一聚合物材料与磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺的质量比为3-7：3-15：3-15，优选为5：10：10；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、所述药物的质量与有机溶剂、水的体积之比为3-7mg：0.5-2.5mL：2-10mL；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、透析之后还包括将脑靶向多肽与含有载药纳米颗粒的悬液混合，超滤去除载药纳米颗粒中游离的脑靶向多肽，获得靶向载药纳米颗粒的步骤；优选的，混合时间为0.5-4h。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.
进一步地，超声的时间为30s以上，例如30s-2min。Furthermore, the ultrasonication time is more than 30 s, for example, 30 s-2 min.
所述活化的NK细胞的制备方法包括，将NK细胞与含巯基类还原剂的溶液混合，孵育，即得；优选的，孵育的温度为30-37℃，时间为10-45min；优选的，每1×106-10×106个细胞中混入0.3-4mg的巯基类还原剂；优选的，含巯基类还原剂的溶液为含巯基类还原剂的PBS溶液；优选的，含巯基类还原剂的溶液中巯基类还原剂的浓度为0.1-0.5mg/mL。The method for preparing 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, 0.3-4 mg of the thiol reducing agent is mixed into every 1×106 -10×106 cells; 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.
本发明还提供了上述任一所述的NK细胞药物递送系统或者上述任一所述的制备方法制得的NK细胞药物递送系统在制备脑部疾病的诊断试剂或者制备预防或治疗脑部疾病的药物中的用途。The present invention also provides use of any of the above-mentioned NK cell drug delivery systems or 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.
1、本发明提供的NK细胞药物递送系统，包括活化的NK细胞和结合在所述活化的NK细胞表面的载药纳米颗粒，所述活化的NK细胞为表面含巯基的NK细胞，所述载药纳米颗粒表面含有马来酰亚胺基团或者巯基基团，所述载药纳米颗粒通过马来酰亚胺基团或者巯基基团与活化的NK细胞表面的巯基相结合，通过NK细胞显著提高了载药纳米颗粒穿越血脑屏障能力进入脑部炎症病灶；此外，NK细胞能够通过识别炎症病灶中的靶标细胞，如感染细胞或炎症细胞，实现特异性细胞治疗；更重要的是NK细胞能够分泌多种细胞因子和介质，调节免疫反应和减轻炎症反应，从而在多个层面上影响疾病的发展和治疗效果，从而产生更强的治疗效果。载药纳米颗粒通过化学偶联在NK细胞表面，通过“搭便车”方式穿越血脑屏障，到达脑内的炎症部位，提高了药物利用率，减少了游离药物对机体的毒副作用。总之，本发明的NK细胞药物递送系统通过马来酰亚胺基团或者巯基基团与巯基相结合的NK细胞和载药纳米颗粒，对脑部疾病细胞或者组织具有高度的靶向性、低生物毒性，显著提高治疗脑部疾病的效果。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 the surface, and the drug-loaded nanoparticles contain maleimide groups or thiol groups on the surface of the activated NK cells, and the drug-loaded nanoparticles are combined with the thiol groups on the surface of the activated NK cells through the maleimide groups or thiol groups, and the drug-loaded nanoparticles are significantly improved through the NK cells. The ability of the drug-loaded nanoparticles to cross the blood-brain barrier enters the brain inflammation focus; in addition, NK cells can achieve specific cell therapy by identifying target cells in the inflammatory focus, 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 the disease and the therapeutic effect at multiple levels, thereby producing a stronger therapeutic effect. The drug-loaded nanoparticles are chemically coupled to the surface of the NK cells, cross the blood-brain barrier by "hitchhiking", reach the inflammatory site in the brain, improve the drug utilization rate, and reduce the toxic and 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 biological toxicity to brain disease cells or tissues through NK cells and drug-loaded nanoparticles that combine maleimide groups or thiol groups with thiol groups, and significantly improves the effect of treating brain diseases.
2、本发明提供的NK细胞药物递送系统，通过优化聚合物载体的原料，即包括第一聚合物材料以及磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺，所述第一聚合物材料选自聚乳酸-羟基乙酸共聚物、聚乳酸、聚己内酯中的至少一种；尤其是以二硬脂酰基磷脂酰乙醇胺-酮缩硫醇-聚乙二醇-马来酰亚胺和聚乳酸-羟基乙酸共聚物（PLGA50k，乳酸：羟基乙酸=50:50）为原料，并且控制所述第一聚合物材料与磷脂-酮缩硫醇-聚乙二醇-马来酰亚胺的质量比为3-15:3-15时（尤其是1：1）使得药物可以高效地负载在聚合物颗粒内，形成稳定的、高包封率、高负载量的载药纳米颗粒，载药纳米颗粒具有较好的生物相容性、生物可降解性；此外，载药纳米颗粒可以在炎症部位的氧化应激（ROS）的作用下，从NK细胞表面脱落释放，更好的发挥作用。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-ketalthioethanol-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-ketalthioethanol-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-ketalthioethanol-polyethylene glycol-maleimide is controlled to be 3-15:3-15 (especially 1:1), the drug can be efficiently loaded in the polymer particles to form stable, high encapsulation rate, and high loading amount of 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, so as to better play a role.
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中：In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:
图1为实施例1制备的载药纳米颗粒RVG-NPs的粒径分布图。横坐标Size为粒径，纵坐标intensity为含量百分数。Figure 1 is a particle size distribution diagram of drug-loaded nanoparticles RVG-NPs prepared in Example 1. The abscissa Size is the particle size, and the ordinate Intensity is the content percentage.
图2为实验例3中有无TCEP处理的情况下，载药纳米颗粒RVG-NPs连接NK细胞的共聚焦荧光成像图。FIG. 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.
图3实验例2中为不同浓度RVG-NPs连接NK细胞的效率的统计图以及荧光成像图。横坐标：RVG-NPs concentration为RVG-NPs的浓度，纵坐标FL intensity为荧光强度。Figure 3 is a statistical diagram and fluorescence imaging diagram of the efficiency of RVG-NPs with different concentrations connecting to NK cells in Experimental Example 2. The horizontal axis: RVG-NPs concentration is the concentration of RVG-NPs, and the vertical axis FL intensity is the fluorescence intensity.
图4为实验例4中通过Transwell实验检测RVG-NPs和RVG-NPs@NKcell处理组穿越体外模拟BBB细胞层，到达Transwell下层培养基和下层细胞的RVG-NPs和RVG-NPs@NK cell的光学显微镜（A）以及荧光显微镜图（B）。Figure 4 shows the optical microscope (A) and fluorescence microscope (B) images of RVG-NPs and RVG-NPs@NK cells in the RVG-NPs and RVG-NPs@NK cell treatment groups detected by the Transwell experiment in Experimental Example 4, which passed through the in vitro simulated BBB cell layer and reached the lower layer of the Transwell culture medium and the lower layer of cells.
图5为实验例4中通过Transwell实验检测RVG-NPs和RVG-NPs@NKcell处理组穿越体外模拟BBB细胞层，到达Transwell下层培养基的NK细胞的计数统计图。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 assay.
图6为实验例4中通过Transwell实验检测RVG-NPs和RVG-NPs@NKcell处理组穿越体外模拟BBB细胞层，到达Transwell下层细胞的RVG-NPs和RVG-NPs@NK cell的荧光统计图。α-syn（+）为加入α-syn蛋白处理组，而α-syn（-）为不加入α-syn蛋白处理组。Figure 6 is a fluorescence statistical graph of RVG-NPs and RVG-NPs@NK cell treated groups passing through the simulated BBB cell layer in vitro and reaching the lower layer of Transwell cells in Experimental Example 4 through the Transwell experiment. α-syn (+) is the group treated with α-syn protein, while α-syn (-) is the group not treated with α-syn protein.
图7为实验例5中在野生型小鼠和帕金森疾病（PD）模型小鼠通过尾静脉注射RVG-NPs@NK cell后9h的脑部荧光成像图（A）和荧光强度统计图（B）。Control为野生型小鼠；PD mice为PD模型小鼠。纵坐标FL intensity为荧光强度。Figure 7 shows brain fluorescence imaging (A) and fluorescence intensity statistics (B) of wild-type mice and Parkinson's disease (PD) model mice 9 hours after injection of RVG-NPs@NK cells through the tail vein in Experimental Example 5. Control is wild-type mice; PD mice are PD model mice. The vertical axis FL intensity is the fluorescence intensity.
图8为实验例6中PD模型小鼠尾静脉注射RVG-NPs和RVG-NPs@NK cell之后不同时间点的脑部荧光成像图。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.
图9为实验例6中PD模型小鼠尾静脉注射RVG-NPs和RVG-NPs@NK cell之后不同时间点的脑部荧光强度统计图。纵坐标Relative Fluorescence intensity为相对荧光强度。Figure 9 is a statistical graph of brain fluorescence intensity at different time points after RVG-NPs and RVG-NPs@NK cells were injected into the tail vein of PD model mice in Experimental Example 6. The vertical axis Relative Fluorescence intensity is the relative fluorescence intensity.
图10为实验例6中PD模型小鼠尾静脉注射RVG-NPs和RVG-NPs@NK cell后24小时主要脏器的荧光成像图。FIG. 10 is a fluorescence imaging image 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.
图11为实验例6中PD模型小鼠尾静脉注射RVG-NPs和RVG-NPs@NK cell后24小时主要脏器中的荧光强度统计图。纵坐标Relative Fluorescence intensity为相对荧光强度。Figure 11 is a statistical graph of the fluorescence intensity in the main organs of PD model mice 24 hours after the 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.
上述附图中，Heart、Liver、Spleen、Lung、Kidney、Brain依次为心脏、肝脏、脾脏、肺脏、肾脏、脑。In the above figures, Heart, Liver, Spleen, Lung, Kidney and Brain represent heart, liver, spleen, lung, kidney and brain respectively.
*表示两组之间的显著性差异p<0.05，**表示p<0.01，***表示p<0.001。* indicates a significant difference between the two groups (p<0.05), ** indicates p<0.01, and *** indicates p<0.001.
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。本发明中，术语ICG为吲哚菁绿。The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. In the present invention, the term ICG is indocyanine green.
实施例1 载药纳米颗粒的制备Example 1 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，包括如下步骤：This embodiment provides a method for preparing drug-loaded nanoparticles, comprising the following steps:
（1）称取模拟药物ICG（5mg）、聚乳酸-羟基乙酸共聚物（PLGA50k，乳酸：羟基乙酸=50:50，10mg）和ROS响应性磷脂（二硬脂酰基磷脂酰乙醇胺-酮缩硫醇-聚乙二醇-马来酰亚胺，DSPE-TK-PEG2k-Mal，10 mg）溶解在1mL的乙醇与二甲基亚砜的混合溶剂（1/9，v/v）中。(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).
（2）在超声条件下（功率：30W，时间：1min），向上述混合溶液中滴加超纯水（5mL）。(2) Under ultrasonic conditions (power: 30 W, time: 1 min), add ultrapure water (5 mL) to the above mixed solution.
（3）上述混合液用超纯水透析（48h）（透析袋MWCO＝3500Da），除去有机溶剂和游离药物，获得载药纳米颗粒（NPs）悬液。(3) The mixed solution was dialyzed against ultrapure water (48 h) (dialysis bag MWCO = 3500 Da) to remove organic solvent and free drug to obtain drug-loaded nanoparticle (NPs) suspension.
（4）将RVG29肽（2μg）加入载药纳米颗粒（NPs）悬液中，混合2h，超滤去除载药纳米颗粒中游离的RVG29肽，获得靶向载药纳米颗粒（RVG-NPs）。(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).
用粒度仪表征载药纳米颗粒的粒径及粒径分布（如图1所示），4℃保存备用。从图1可以看出，RVG-NPs的粒径分布在80-130nm之间，其中平均粒径为105 nm。PDI多分散系数是0.2±0.1。The particle size and particle size distribution of the drug-loaded nanoparticles were characterized by a particle size meter (as shown in Figure 1) and stored at 4°C for future use. As can be seen from Figure 1, the particle size distribution of RVG-NPs is between 80-130 nm, with an average particle size of 105 nm. The PDI polydispersity coefficient is 0.2±0.1.
实施例2 载药纳米颗粒的制备Example 2 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，包括如下步骤：This embodiment provides a method for preparing drug-loaded nanoparticles, comprising the following steps:
将白蛋白（HSA）溶于水中配成5mL浓度为0.375mg/mL的水溶液，得到水相；然后将5mg的ICG溶解在1mL二氯甲烷/乙醇（体积比9:1）的混合溶液中，得到油相；将油相滴加到水相中，边滴加边剪切2min，并通过薄膜蒸发法去除有机溶剂，获得载药纳米颗粒。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.
实施例3 载药纳米颗粒的制备Example 3 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，基本与实施例1相同，区别仅在于：步骤（1）中模拟药物ICG的质量由5mg调整为2mg。This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of Embodiment 1, except that the mass of the simulated drug ICG in step (1) is adjusted from 5 mg to 2 mg.
实施例4 载药纳米颗粒的制备Example 4 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，基本与实施例1相同，区别仅在于：步骤（1）中ROS响应性磷脂的质量由10mg调整为3mg。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.
实施例5 载药纳米颗粒的制备Example 5 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，基本与实施例1相同，区别仅在于：步骤（1）中聚乳酸-羟基乙酸共聚物的质量由10mg调整为3mg。This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of Embodiment 1, except that the mass of the polylactic acid-glycolic acid copolymer in step (1) is adjusted from 10 mg to 3 mg.
实施例6 载药纳米颗粒的制备Example 6 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，基本与实施例1相同，区别仅在于：步骤（2）的超声功率由30W调整为10W。经检测制得的RVG-NPs的粒径分布为165-205nm之间。This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of embodiment 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 between 165 and 205 nm.
实施例7 载药纳米颗粒的制备Example 7 Preparation of drug-loaded nanoparticles
本实施例提供了一种载药纳米颗粒的制备方法，基本与实施例1相同，区别仅在于：步骤（2）的超声功率由30W调整为20W。经检测制得的RVG-NPs的粒径分布为140-180nm之间。This embodiment provides a method for preparing drug-loaded nanoparticles, which is basically the same as that of embodiment 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 between 140 and 180 nm.
实施例8NK细胞药物递送系统的制备Example 8 Preparation ofNK cell drug delivery system
本实施例提供了一种NK细胞药物递送系统的制备方法，包括如下步骤：This embodiment provides a method for preparing a NK cell drug delivery system, comprising the following steps:
（1）将NK细胞（2mL，细胞总数2×106个）加入到含有三（2-羧乙基）-膦盐酸盐（TCEP，浓度：0.3mg/mL）的PBS（5mL）中，在37°C下孵育30 min，再用PBS洗涤3次，最后将TCEP处理后的NK细胞分散到0.1mL的PBS中。(1) Add NK cells (2 mL, total number of cells 2 × 106 ) into PBS (5 mL) containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP, concentration: 0.3 mg/mL), incubate at 37°C for 30 min, wash three times with PBS, and finally disperse the TCEP-treated NK cells into 0.1 mL of PBS.
（2）在上述TCEP处理过的体积为0.1mL的NK细胞中，加入体积为0.1mL、浓度为1.25mg/mL的载药纳米颗粒RVG-NPs（实施例1方法制得）的PBS分散液并混合，在37°C下孵育30min，用PBS洗涤NK细胞药物递送系统3次，除去未连接的载药纳米颗粒，得到NK细胞药物递送系统（RVG-NPs@NK cell）。(2) To the above TCEP-treated NK cells (volume 0.1 mL), a PBS dispersion of drug-loaded nanoparticles RVG-NPs (prepared by the method in Example 1) (volume 0.1 mL) with a concentration of 1.25 mg/mL was added and mixed, and 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 a NK cell drug delivery system (RVG-NPs@NK cell).
实验例1Experimental Example 1
测试载药纳米颗粒的粒径、载药量和包封率。载药量和包封率的具体测试方法如下：载药纳米颗粒经冷冻干燥后，称重，再将其溶于二甲基亚砜（DMSO）。通过紫外分光光度计测量药物在最大吸收峰处的吸光度值，按照下述公式计算载药纳米颗粒的载药量和包封率。载药量和包封率结果如下表1所示。The particle size, drug loading and encapsulation efficiency of the drug-loaded nanoparticles were tested. The specific test methods for drug loading and encapsulation efficiency are as follows: After freeze-drying, the drug-loaded nanoparticles were weighed and then dissolved in dimethyl sulfoxide (DMSO). The absorbance value of the drug at the maximum absorption peak was measured by an ultraviolet spectrophotometer, and the drug loading and encapsulation efficiency of the drug-loaded nanoparticles were calculated according to the following formula. The drug loading and encapsulation efficiency results are shown in Table 1 below.
表1 各实施例载药纳米颗粒的表征结果Table 1 Characterization results of drug-loaded nanoparticles in various examples
将实施例2与实施例1、实施例3-5相比，在投药量相同的情况下，相比于HSA为原料制得的载药纳米颗粒来说，采用PLGA50k/DSPE-TK-PEG2k-Mal制成的聚合物载药纳米颗粒的包封率和载药量明显提高。由实施例1和实施例3-5相比较可知，采用实施例1通过控制药物ICG、聚乳酸-羟基乙酸共聚物和ROS响应性磷脂的质量比得到的聚合物载药纳米颗粒的尺寸恰当，载药量最高。由实施例1，实施例6-7比较可知，超声功率对于聚合物载药纳米颗粒的尺寸影响不大，10W-30W下均可得到粒径分布均匀的载药纳米颗粒。Comparing Example 2 with Example 1 and Examples 3-5, under the same dosage, the encapsulation efficiency and drug loading of polymer drug-loaded nanoparticles made of PLGA50k/DSPE-TK-PEG2k-Mal are significantly improved compared to drug-loaded nanoparticles made of HSA as raw material. By comparing Example 1 with Examples 3-5, it can be seen that the size of polymer drug-loaded nanoparticles obtained by controlling the mass ratio of 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 polymer drug-loaded nanoparticles, and drug-loaded nanoparticles with uniform particle size distribution can be obtained under 10W-30W.
实验例2 荧光强度Experimental Example 2 Fluorescence Intensity
考察不同的载药纳米颗粒浓度（或者药物量）对制备NK细胞药物递送系统的影响。在NK细胞药物递送系统的制备过程中，分别加入1mL的PBS溶液或者1mL浓度分别为31.25、62.5、125或者162.5 μg/mL的载药纳米颗粒RVG-NPs的PBS分散液，其余工艺和条件均按照实施例8的方法制备NK细胞药物递送系统。将实施例8构建的NK细胞药物递送系统（RVG-NPs@NK cell）加入24孔板中，使用小动物成像检测其荧光强度来计算负载效率（如图3所示）。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 with concentrations of 31.25, 62.5, 125 or 162.5 μg/mL was added, and the remaining processes and conditions were prepared according to the method of Example 8. The NK cell drug delivery system (RVG-NPs@NK cell) constructed in Example 8 was added to a 24-well plate, and its fluorescence intensity was detected using small animal imaging to calculate the loading efficiency (as shown in Figure 3).
图3为实施例8制备的NK细胞药物负载效率的统计结果。从图3我们可以看出其最大负载效率在62.5 µg/106细胞，荧光强度约为1.0×107。Figure 3 is the statistical result of the drug loading efficiency of NK cells prepared in Example 8. From Figure 3 we can see that its maximum loading efficiency is 62.5 µg/106 cells, and the fluorescence intensity is about 1.0×107 .
实验例3 形态分析Experimental Example 3 Morphological Analysis
1、实验方法1. Experimental methods
将NK细胞随机分为两组，TCEP+组和TCEP-组。TCEP+组为取NK细胞按照实施例8方法经过TCEP处理后，与载药纳米颗粒RVG-NPs共混得到NK细胞药物递送系统。而TCEP-组为取NK细胞未经过TCEP处理，直接与载药纳米颗粒RVG-NPs共混得到的实验结果，即与实施例8区别仅在于省略步骤（1）。然后向上述NK细胞中加入4%的多聚甲醛固定，并加入DAPI对NK细胞核进行染色。用共聚焦显微镜观察两组NK细胞形态变化及NK细胞表面接枝载药纳米颗粒的效率（如图2所示）。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, that is, 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 surface of NK cells were observed using a confocal microscope (as shown in Figure 2).
2、实验结果2. Experimental results
图2为实施例8的共聚焦显微镜的观察结果。蓝色荧光来源于NK细胞核，红色点状荧光来源于RVG-NPs。从图2可以看出，经过TCEP处理的NK细胞的实验组中，RVG-NPs点状荧光基本都分布在NK细胞膜表面（白色箭头所示），证明利用TCEP处理的方法能能够将RVG-NPs有效地接枝在NK细胞的表面；而未经TCEP处理的NK细胞的对照组中，多数RVG-NPs点状荧光分布在细胞浆中，表明RVG-NPs都被NK细胞摄取并进入细胞浆中。以上实验结果证明了NK细胞药物递送系统的成功构建。Figure 2 is the observation result of confocal microscope in Example 8. The blue fluorescence comes from the NK cell nucleus, and the red dot fluorescence comes from RVG-NPs. As can be seen from Figure 2, in the experimental group of NK cells treated with TCEP, the RVG-NPs dot fluorescence is basically distributed on the surface of the NK cell membrane (indicated by the white arrow), proving that the method of TCEP treatment 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 dot fluorescence is distributed in the cytoplasm, indicating that 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.
实验例4 NK体外迁移实验Experimental Example 4 NK in vitro migration assay
1、实验方法1. Experimental methods
构建体外BBB和模拟PD病灶的实验模型：首先在Transwell小室里面铺上10万个bEnd.3细胞（小鼠脑微血管内皮细胞），培养至细胞紧密贴合。在放置Transwell小室的下层深孔24孔板中铺上10万个BV2细胞（小鼠小胶质细胞）和10万个MN9D细胞（小鼠中脑多巴胺能神经元细胞），待细胞贴壁后向Transwell小室下层培养基中加入α-syn蛋白（即α-突触核蛋白，帕金森病（PD）的病理标志之一是在脑中积累α-突触核蛋白）。继续将Transwell小室放入下层深孔24孔板中，培养24 h。在Transwell小室中，分别加入30μL实施例8制得的NK细胞药物递送系统（RVG-NPs@NK cell）或者30μL浓度为1.25mg/mL的载药纳米颗粒RVG-NPs（实施例1方法制得）的PBS分散液。以不加入α-syn蛋白只加RVG-NPs@NK cell作为对照，相同条件下处理。6h后分离下层的上清液和下层细胞。在上清液和下层细胞中加入多聚赖氨酸固定细胞，1h后用光学显微镜观察和统计上清液中穿过体外BBB的NK细胞的数量；用DAPI对下层细胞核进行染色，然后用荧光显微镜观察下层细胞和统计下层细胞中RVG-NPs的荧光强度。Construct an experimental model of in vitro BBB and simulated PD lesions: First, 100,000 bEnd.3 cells (mouse brain microvascular endothelial cells) were placed in the Transwell chamber and cultured until the cells were tightly attached. 100,000 BV2 cells (mouse microglia) and 100,000 MN9D cells (mouse midbrain dopaminergic neuron cells) were placed in the lower deep-well 24-well plate where the Transwell chamber was placed. After the cells adhered to the wall, α-syn protein (i.e., α-synuclein, one of the pathological signs of Parkinson's disease (PD) is the accumulation of α-synuclein in the brain) was added to the culture medium in the lower layer of the Transwell chamber. Continue to place the Transwell chamber in the lower deep-well 24-well plate and culture for 24 h. In the Transwell chamber, 30 μL of the NK cell drug delivery system (RVG-NPs@NK cell) prepared in Example 8 or 30 μL of PBS dispersion of drug-loaded nanoparticles RVG-NPs (prepared by the method of Example 1) with a concentration of 1.25 mg/mL were added. The control group was treated with RVG-NPs@NK cells without α-syn protein. The supernatant and cells in the lower layer were separated after 6 hours. Poly-lysine was added to the supernatant and cells in the lower layer to fix the cells. After 1 hour, the number of NK cells that passed through the BBB in vitro 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 then the cells in the lower layer were observed and the fluorescence intensity of RVG-NPs in the cells in the lower layer was counted using a fluorescence microscope.
2、实验结果2. Experimental results
从图4A和图5结果可以看出，在α-syn存在的条件下，NK细胞的穿越效率显著提升，证明了NK细胞药物递送系统具有靶向PD病灶或炎症部位归巢的能力。从图4B和图6结果可以看出，NK细胞药物递送系统处理组的下层细胞（BV2细胞和MN9D细胞）胞浆内的红色荧光信号（来源于RVG-NPs）明显增加，表明NK细胞药物递送系统可以有效地携带载药纳米颗粒穿越体外BBB；同时，载药纳米颗粒进入PD病灶部位后，能从NK细胞表面脱落，最后被靶细胞（BV2细胞和MN9D细胞）摄取。As can be seen from the results in Figures 4A and 5, the NK cell crossing efficiency is significantly improved in the presence of α-syn, proving that the NK cell drug delivery system has the ability to target PD lesions or inflammatory sites. As can be seen from the results in Figures 4B and 6, the red fluorescence signal (derived from RVG-NPs) in the cytoplasm of the lower cells (BV2 cells and MN9D cells) in the NK cell drug delivery system treatment group increased significantly, indicating that the NK cell drug delivery system can effectively carry drug-loaded nanoparticles across the BBB in vitro; at the same time, after entering the PD lesion site, the drug-loaded nanoparticles can fall off from the surface of NK cells and finally be taken up by the target cells (BV2 cells and MN9D cells).
实验例5 NK体内靶向脑部病灶实验Experimental Example 5 NK targeting brain lesions in vivo
1、实验方法1. Experimental methods
将6只C57小鼠按性别及体重随机分为实验对照组（Control）和帕金森疾病模型组（PD mice组或者PD模型组），每组3只。对于PD模型组的小鼠，通过脑定位注射AAV-A53T α-Syn病毒（3个TCID50，0.5μL）；对于实验对照组的小鼠，通过脑定位注射对照病毒（AAV，0.5μL），并在肌肉中注射青霉素。病毒注射完1个月后，小鼠帕金森疾病模型构建成功。分别在实验对照组和帕金森疾病模型组小鼠的尾静脉中注射CM-DiI标记的NK细胞（注射剂量为2×106个，200μL），在9小时后取出小鼠的脑部进行荧光成像及荧光强度分析。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 mice in the PD model group, AAV-A53T α-Syn virus (3 TCID50, 0.5μL) was injected through brain localization; for mice in the experimental control group, control virus (AAV, 0.5μL) 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 2×106 , 200μL) were injected into the tail vein of mice in the experimental control group and the Parkinson's disease model group, respectively, and the brains of the mice were removed 9 hours later for fluorescence imaging and fluorescence intensity analysis.
2、实验结果2. Experimental results
结果见图7所示。从图7的荧光成像图和荧光强度统计结果可以看出，相对于实验对照组小鼠来说，PD模型组的脑组织荧光强度更高，表明更多的NK细胞富集在PD模型鼠的脑组织或蓄积在具有炎症的脑部病灶部位。The results are shown in Figure 7. From the fluorescence imaging diagram and fluorescence intensity statistical results in Figure 7, it can be seen that compared with the experimental control group mice, the brain tissue fluorescence intensity of the PD model group is higher, indicating that more NK cells are enriched in the brain tissue of the PD model mice or accumulated in the brain lesions with inflammation.
实验例6 NK细胞药物递送系统体内靶向脑部病灶实验Experimental Example 6 In vivo targeting of brain lesions by NK cell drug delivery system
1、实验方法1. Experimental methods
按照实验例5的方法构建帕金森疾病（PD）模型小鼠。将PD模型小鼠随机分为载药纳米颗粒（RVG-NPs）组和NK细胞药物递送系统（RVG-NPs@NK cell）组。对于RVG-NPs组，通过尾静脉注射给予RVG-NPs的悬液（载药纳米颗粒浓度为1.25mg/mL）；对于RVG-NPs@NK cell组，通过尾静脉注射给予实施例8制得的RVG-NPs@NK cell，给药体积均为150μL。分别在1，3，6，9，12，24小时通过小动物成像仪观察和检测脑组织部位的荧光强度。在24小时取出主要脏器对其进行荧光成像及荧光强度分析。Parkinson's disease (PD) model mice were constructed 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. For the RVG-NPs group, a suspension of RVG-NPs (drug-loaded nanoparticle concentration of 1.25 mg/mL) was administered by tail vein injection; for the RVG-NPs@NK cell group, the RVG-NPs@NK cell prepared in Example 8 was administered by tail vein injection, and the administration volume was 150 μL. The fluorescence intensity of the brain tissue was observed and detected by a small animal imager at 1, 3, 6, 9, 12, and 24 hours, respectively. The main organs were removed at 24 hours for fluorescence imaging and fluorescence intensity analysis.
2、实验结果2. Experimental results
从图8-9的结果可以看出，NK细胞药物递送系统脑内蓄积效率明显高于载药纳米颗粒组，证明了NK细胞药物递送系统能够在脑内炎症部位高效蓄积。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 accumulate efficiently in inflammatory sites in the brain.
从图10-11的结果可以看出，NK细胞药物递送系统和载药纳米颗粒组在主要脏器中的分布没有显著性差异，但是NK细胞药物递送系统在脑内的荧光强度为3.3×108，载药纳米颗粒在脑内的荧光强度为1.1×108，NK细胞药物递送系统脑内的蓄积效率约为载药纳米颗粒的3倍，证明利用NK细胞药物递送策略能显著提升药物在脑内的蓄集效率，有望提升脑部疾病的治疗效果。From the results of Figures 10-11, it can be seen that there is no significant difference in the distribution of the NK cell drug delivery system and the drug-loaded nanoparticle group in the major organs. However, the fluorescence intensity of the NK cell drug delivery system in the brain is 3.3×108 , and the fluorescence intensity of the drug-loaded nanoparticles in the brain is 1.1×108 . The accumulation efficiency of the NK cell drug delivery system in the brain is about 3 times that of the 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 therapeutic effect of brain diseases.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.
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