WO2022198742A1 - 一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法 - Google Patents

一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法 Download PDF

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WO2022198742A1
WO2022198742A1 PCT/CN2021/089946 CN2021089946W WO2022198742A1 WO 2022198742 A1 WO2022198742 A1 WO 2022198742A1 CN 2021089946 W CN2021089946 W CN 2021089946W WO 2022198742 A1 WO2022198742 A1 WO 2022198742A1
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preparation
exosome
superparamagnetic
vesicle
drug
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French (fr)
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张徐
张家慧
纪成
史惠
许文荣
钱晖
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江苏大学
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
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    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications
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Definitions

  • the invention belongs to the technical field of an engineered nanovesicle drug-carrying system, in particular to a superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation and a preparation method thereof.
  • Cancer is a disease that seriously threatens human health, and most cancers are diagnosed in the middle and late stages, resulting in poor prognosis for patients.
  • chemotherapy is a common means of treating cancer.
  • most chemotherapeutic drugs have toxic effects on normal cells and lack targeting. Therefore, the emergence of extracellular vesicles opens up new prospects for tumor-targeted therapy. From the perspective of natural cell-derived nanocarriers, extracellular vesicles have low biological toxicity and can be used as excellent nanocarriers to be designed into complexes with targeted antitumor effects.
  • the purpose of the present invention is to provide a superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation and a preparation method thereof, the superparamagnetic modified neutrophil exosome biomimetic vesicle Bubble drug delivery biologics can specifically target cancer cells, thereby inhibiting cancer cell proliferation and delaying the occurrence and development of cancer.
  • the present invention provides a method for preparing a superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation, which comprises the following steps: 1) co-culturing peripheral blood neutrophils with antitumor drugs, and obtaining uptake drug neutrophils;
  • the gradient filtration includes sequentially passing through 1 ⁇ m, 400 nm and 200 nm polycarbonate filter membranes ;
  • each 1 ⁇ 10 6 peripheral blood neutrophils are co-cultured with 50 ⁇ g of an antitumor drug.
  • the temperature of the co-cultivation in step 1) is 37°C, and the time is 12-24 hours.
  • the centrifugation in step 2) includes differential centrifugation, the centrifugal force of the differential centrifugation is 800 g, and the centrifugation time is 5 minutes.
  • the physical extrusion in step 3) includes 11 consecutive physical extrusions on the cell suspension using a mini-extrader extruder.
  • the centrifugation in step 4) includes ultracentrifugation, and the centrifugal force of the ultracentrifugation is 10,000 g and the centrifugation time is 80 minutes.
  • the superparamagnetic material in step 5) includes SPION-Tf, and the concentration of the SPION-Tf in the NNV-drug suspension is 0.5 mg/ml.
  • the temperature of the co-incubation in step 5) is 4°C and the time is 4h.
  • the present invention also provides the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation prepared by the above preparation method.
  • the invention provides a preparation method of superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation.
  • a mini-extrader extruder is used to extrude DOX-phagocytic neutrophils through 1 ⁇ m, 400 nm and 200 nm polymerization. After extrusion of the carbonate filter membrane, neutrophil exosome biomimetic vesicles with different particle sizes were formed to deliver biologics.
  • the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation prepared by the preparation method of the present invention acts on tumor cell lines, and it is found that the superparamagnetic modified neutrophil
  • the cellular exosome biomimetic vesicle drug delivery biologics can specifically target tumor cells, induce tumor cell apoptosis, inhibit tumor growth, significantly prolong the survival time of mice, and improve the therapeutic efficiency of drugs against cancer.
  • Fig. 1 is the flow chart of the preparation method of superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation in Example 1;
  • Figure 2 shows the uptake of adriamycin liposomes by human peripheral blood neutrophils in Example 1;
  • Fig. 3 is the preparation and transmission electron microscope picture of the biological preparation of human peripheral blood neutrophil exosome biomimetic vesicles encapsulating medicine in Example 1;
  • Fig. 4 is the particle size and zeta potential of the biological preparation of human peripheral blood neutrophil exosome biomimetic vesicles encapsulating the drug in Example 1;
  • Figure 5 is the cytotoxic effect of the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation on tumor cell lines in Example 1, wherein a is the in vitro co-existence of HGC-27 cells and SPION-NNV-DOX/MF Culture pattern diagram; b is the proliferation ability of HGC-27 cells under different treatments detected by CCK8 cell proliferation assay.
  • the present invention provides a method for preparing a superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation, which comprises the following steps: 1) co-culturing peripheral blood neutrophils with antitumor drugs, and obtaining uptake drug neutrophils;
  • the gradient filtration includes sequentially passing through 1 ⁇ m, 400 nm and 200 nm polycarbonate filter membranes ;
  • peripheral blood neutrophils and antitumor drugs are co-cultured to obtain neutrophils that take in drugs.
  • each 1 ⁇ 10 6 peripheral blood neutrophils are preferably co-cultured with 50 ⁇ g of antitumor drugs.
  • the present invention does not specifically limit the sources of the peripheral blood neutrophils and anti-tumor drugs, and the conventional extraction methods in the art or commercially available products may be used.
  • the antitumor drug of the present invention preferably comprises doxorubicin liposome (DOX-CL).
  • human peripheral blood neutrophils are preferably cultured for 6 hours with a medium RPMI1640 containing 10% fetal bovine serum, and then a solution of antitumor drugs is added to the culture medium for co-cultivation for 24 hours.
  • the temperature of the co-cultivation is preferably 37°C.
  • the time is preferably 12 to 24 hours.
  • the cytoplasm of neutrophils contained a large amount of antitumor drugs (DOX) with red fluorescence.
  • DOX antitumor drugs
  • the present invention centrifuges the neutrophils taking the medicine, and resuspends the bottom buffer to obtain a cell suspension.
  • the centrifugation in the present invention preferably includes differential centrifugation, the centrifugal force of the differential centrifugation is preferably 800 g, and the centrifugation time is preferably 5 min.
  • the buffer at the bottom is preferably collected, and the pellet is resuspended with PBS to prepare a cell suspension of neutrophil-coated drug.
  • the present invention performs continuous physical extrusion and gradient filtration on the cell suspension to obtain a neutrophil exosome biomimetic vesicle-wrapped drug suspension; the gradient filtration includes successively passing 1 ⁇ m, 400 nm and 200nm polycarbonate filter membrane.
  • the physical extrusion of the present invention preferably includes using a mini-extrader extruder (Avanti Polar Lipids, USA) to perform 11 consecutive physical extrusions on the cell suspension, and the parameters of the physical extrusion preferably include: 5 mL of cells
  • the extrusion parameters of the suspension are: 300 psig, and the temperature of the air-tight syringe is above 10°C.
  • the present invention can generate neutrophil exosome biomimetic vesicles with different particle sizes through physical extrusion, and can simply and quickly prepare a large number of neutrophil exosome biomimetic vesicles.
  • the physically extruded cell suspension is sequentially passed through 1 ⁇ m, 400 nm and 200 nm polycarbonate filter membranes, and the extruded filtrate is collected to prepare a neutrophil exosome biomimetic vesicle-coated drug suspension. liquid.
  • the present invention centrifuges the neutrophil exosome biomimetic vesicle-wrapped drug suspension, and resuspends the bottom NNV-drug buffer to obtain NNV -Drug suspension.
  • the centrifugation in the present invention preferably includes ultracentrifugation, the centrifugal force of the ultracentrifugation is preferably 10000g, and the centrifugation time is preferably 80min.
  • the resuspension of the present invention preferably includes collecting the NNV-drug buffer at the bottom after the ultracentrifugation, and redispersing the NNV-drug precipitate in a PBS buffer for further purification.
  • the NNV-drug suspension is mixed with the superparamagnetic material and then incubated together, and the mixed solution after the co-incubation is magnetically separated to obtain the superparamagnetic modified neutrophil exocytosis.
  • the superparamagnetic material of the present invention preferably includes SPION-Tf, and the concentration of the SPION-Tf in the NNV-drug suspension is 0.5 mg/ml.
  • the temperature of the co-incubation in the present invention is preferably 4°C, and the time is preferably 4h.
  • the present invention also provides the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation prepared by the above preparation method.
  • the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation is a neutrophil extruded vesicle encapsulated with doxorubicin liposome (DOX-CL) and SPION - Cup-shaped membrane-shaped vesicles with a particle size of 200 nm obtained by magnetic separation after Tf binding;
  • the active ingredients of the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation include neutrophil-related Toxic protein (FasL, GranzymeA/B, Perforin);
  • the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation plays a double killing effect in gastric cancer cells by carrying the toxic protein and the chemotherapeutic drug DOX, Significantly inhibited tumor growth (15%
  • Human peripheral blood neutrophil culture reagents polymorphonuclear leukocyte separation medium (PolymorphPrep separation medium, Norway), RPMI1640 (Bioind, USA), fetal bovine serum (Gibco, USA), trypsin (Sigma, USA), carbon dioxide culture box (Forma company), serum-free medium (Excell, China);
  • Extraction reagents for SPION-NNV-DOX biological preparations SPION material and doxorubicin liposomes (Xian ruixi Biological Technology, China), Mini-extrader extruder (Avanti PolarLipids, USA), 1 ⁇ m, 400nm, 200nm different pore sizes Polycarbonate filter membrane (Xian ruixi Biological Technology, China), transmission electron microscope (FEI Tecnai 12, Philips), ultracentrifuge (Beckman, USA), panoramic flow cytometer (Flow sight, USA), NanoSight LM10 system (nanosight tracking analysis, UK).
  • the superparamagnetic modified neutrophil exosome biomimetic vesicle drug delivery biological preparation was prepared according to the process shown in Figure 1:
  • the cell suspension was continuously extruded 11 times using a mini-extrader extruder, and then passed through 1 ⁇ m, 400 nm, and 200 nm polycarbonate filter membranes to prepare DOX (NNV- DOX, NNV: Neutrophil Nano Vesicle, neutrophil extruded vesicle);
  • NNV-DOX suspension was incubated with SPION-Tf (0.5mg/ml) superparamagnetic material, 4°C, 4h;
  • NNV-DOX Nanoparticle Tracking Analysis
  • 96-well cell culture plate JET Biofil
  • RPMI1640 Bioind, USA
  • trypsin Sigma, USA
  • CCK8 detection kit Vazyme, China
  • microplate reader FLX800, United States

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Abstract

一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法，其特征在于，制备方法包括以下步骤： 1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞； 2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液； 3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜； 4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液； 5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。

Description

一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法
本申请要求于2021年03月26日提交中国专利局、申请号为202110324298.1、发明名称为“一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于工程化纳米囊泡载药系统技术领域，具体涉及一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法。
背景技术
癌症是严重威胁人类健康的疾病，大多数癌症的诊断都处于中晚期，导致患者预后不佳。目前，化疗是治疗癌症的常用手段。然而，大多数化疗药物对正常细胞有毒性作用，缺乏靶向性。因此，细胞外囊泡的出现为肿瘤的靶向治疗开辟了新前景。从天然细胞来源的纳米载体来看，细胞外囊泡具有较低的生物毒性，可以作为优良的纳米载体被设计成具有靶向抗肿瘤作用的复合物。迄今为止，已有诸多研究通过细胞外囊泡输送蛋白、RNA或其他小分子药物以达到治疗疾病的目的，但是中性粒细胞外泌体的仿生囊泡未曾在肿瘤领域进行过相关研究。
发明内容
有鉴于此，本发明的目的在于提供一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂可特异性靶向癌细胞，从而抑制癌细胞增殖、延缓癌发生发展。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，包括以下步骤：1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
优选的，步骤1)每1×10 6个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。
优选的，步骤1)所述共同培养的温度为37℃，时间为12～24h。
优选的，步骤2)所述离心包括差速离心，所述差速离心的离心力为800g，离心时间为5min。
优选的，步骤3)所述物理挤压包括使用mini-extrader挤压器对所述细胞悬液进行11次连续的物理挤压。
优选的，步骤4)所述离心包括超速离心，所述超速离心的离心力为10000g，离心时间为80min。
优选的，步骤5)所述超顺磁材料包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。
优选的，步骤5)所述共孵育的温度为4℃，时间为4h。
优选的，步骤5)所述磁分离包括利用钕磁铁进行磁分离，MF＝1T。
本发明还提供了利用上述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送 生物制剂的制备方法，利用mini-extrader挤压器将吞噬DOX的中性粒细胞通过1μm、400nm和200nm聚碳酸酯过滤膜挤压后，形成不同粒径的中性粒细胞外泌体仿生囊泡药物递送生物制剂。
在本发明实施例中，利用本发明所述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂，作用于肿瘤细胞株，发现超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂具有特异性靶向肿瘤细胞，诱导肿瘤细胞凋亡，抑制肿瘤生长，显著延长小鼠生存时间，改善药物对癌症的治疗效率。
附图说明
图1为实施例1中超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法流程图；
图2为实施例1中人外周血中性粒细胞对阿霉素脂质体的摄取情况；
图3为实施例1中人外周血中性粒细胞外泌体仿生囊泡包裹药物的生物制剂的制备和透射电镜图；
图4为实施例1中人外周血中性粒细胞外泌体仿生囊泡包裹药物的生物制剂的粒径大小和zeta电位；
图5为实施例1中超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂对肿瘤细胞株的细胞毒性作用，其中a为HGC-27细胞与SPION-NNV-DOX/MF体外共培养模式图；b为CCK8细胞增殖实验检测不同处理下HGC-27细胞增殖能力情况。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，包括以下步骤：1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
本发明将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞。本发明优选每1×10 6个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。本发明对所述外周血中性粒细胞和抗肿瘤药物的来源并没有特殊限定，利用本领域的常规提取方法提取或市售产品即可。本发明所述抗肿瘤药物优选包括阿霉素脂质体(DOX-CL)。本发明优选用含10％胎牛血清的培养基RPMI1640培养人外周血中性粒细胞6h，然后添加抗肿瘤药物的溶液至培养液中共同培养24h，所述共同培养的温度优选为37℃，时间优选为12～24h。经过所述共同培养后，中性粒细胞胞质内含有大量带有红色荧光的抗肿瘤药物(DOX)。
得摄取药物的中性粒细胞后，本发明对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液。本发明所述离心优选包括差速离心，所述差速离心的离心力优选为800g，离心时间优选为5min。本发明经所述离心后，优选收集底部的缓冲垫，用PBS再次重悬沉淀制备中性粒细胞包裹药物的细胞悬液。
得细胞悬液后，本发明对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜。本发明所述物理挤压优选包括使用mini-extrader挤压器(Avanti Polar Lipids,USA)对所述细胞悬液进行11次连续的物理挤压，所述物理挤压的参数优选包括：5mL细胞悬液挤压参数为：300psig，气密注射器温度为10℃以上。本发明通过物理挤压可以产生不同粒径大小的中性粒细胞外泌体仿生囊泡，可以简单快速制备出大量中性粒细胞外泌体仿生囊泡。本发明使经过所述物理挤压的细胞悬液依次通过1μm、400nm、200nm聚碳酸酯过滤膜，收集挤压后的滤过液，制备得到中性粒细胞外泌体仿生囊泡包裹药物悬液。
得中性粒细胞外泌体仿生囊泡包裹药物悬液后，本发明对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液。本发明所述离心优选包括超速离心，所述超速离心的离心力优选为10000g，离心时间优选为80min。本发明所述重悬优选包括收集经所述超速离心后底部的NNV-药物缓冲垫，将NNV-药物沉淀再分散于PBS缓冲液中进一步纯化。
得NNV-药物悬液后，本发明将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。本发明所述超顺磁材料优选包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。本发明所述共孵育的温度优选为4℃，时间优选为4h。本发明对共孵育后的混合液进行磁分离，所述磁分离优选包括利用钕磁铁进行磁分离，MF＝1T。
本发明还提供了利用上述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。本发明实施例中，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂是包裹阿霉素脂质体(DOX-CL)的中性粒细胞挤压囊泡与SPION-Tf结合后磁分离得到的粒径在200nm的杯状膜形囊泡；所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的活性成份包括中性粒细胞相关的毒性蛋白(FasL,GranzymeA/B,Perforin)；所述的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂通过运载毒性蛋白和化疗药物DOX在胃癌细胞中发挥双重杀伤作用，显著抑制肿瘤生长(载药量为15％)。
下面结合实施例对本发明提供的一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂提取与鉴定
人外周血中性粒细胞培养试剂：多形核白细胞分离液(PolymorphPrep分离液，Norway)、RPMI1640(Bioind，USA)、胎牛血清(Gibco， USA)、胰蛋白酶(Sigma，USA)、二氧化碳培养箱(Forma公司)、无血清培养基(Excell，China)；
倒置显微镜(Nikon，Japan)、超净工作台，台式离心机(Eppendorf，Germany)，超速离心机(Beckman，USA)。
SPION-NNV-DOX生物制剂的提取试剂：SPION材料和阿霉素脂质体(Xi an ruixi Biological Technology，China)、Mini-extrader挤压器(Avanti PolarLipids，USA)、1μm，400nm，200nm不同孔径的聚碳酸酯过滤膜(Xi an ruixi Biological Technology，China)、透射电子显微镜(FEI Tecnai 12，Philips公司)、超速离心机(Beckman，USA)、全景式流式细胞仪(Flow sight，USA)、NanoSight LM10 system(nanosight tracking analysis，UK)。
按照图1所示流程制备超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂：
1.将人外周血中性粒细胞(Neu，Neutrophils，中性粒细胞)与阿霉素脂质体(DOX-CL)在孵箱内37℃共培养24h(图2)；
2.800g，5min(Eppendorf Centrifuge 5804/5804离心机)离心，收集底部的缓冲垫，用PBS冲悬制备细胞悬液；
3.使用mini-extrader挤压器对细胞悬液进行连续挤压11次，依次通过1μm，400nm，200nm聚碳酸酯过滤膜，制备中性粒细胞外泌体仿生囊泡包裹的DOX(NNV-DOX，NNV：Neutrophil Nano Vesicle，中性粒细胞挤压囊泡)；
4.通过超速离心法：10000g，80min，(Beckman超速离心机)离心沉淀收集NNV-DOX，将NNV-DOX沉淀再分散于PBS中；
5.NNV-DOX悬液与SPION-Tf(0.5mg/ml)超顺磁材料共同孵育，4℃，4h；
6.通过外加钕磁铁(MF＝1T)对混合液进行磁分离获得SPION-NNV-DOX复合物。
透射电镜和Nanoparticle Tracking Analysis观察(NNV-DOX)形态特征：阿霉素脂质体、中性粒细胞挤压囊泡和NNV-DOX溶液各20μL，混匀后滴加于直径2mm的载样铜网上，静置5min后，用滤纸吸去残余液体，将铜网倒扣于30g/L磷钨酸(pH 6.8)液滴上，25℃条件下负染5min，白 炽灯下烘干，透射电镜下观察拍照，图3所示阿霉素脂质体的直径约为50nm，中性粒细胞挤压囊泡为大小在180nm左右的囊泡状结构；NNV-DOX的粒径约为220nm左右；图4显示NNV-DOX的粒径大小为200nm±20左右，电位为-29.04±0.45mV。
实施例2
超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂对肿瘤细胞的体外抗肿瘤作用
96孔细胞培养板(JET Biofil公司)，RPMI1640(Bioind，USA)、胰蛋白酶(Sigma，USA)、CCK8检测试剂盒(Vazyme，China)、酶标仪(FLX800，United States)。
1.用胰蛋白酶消化HGC-27细胞，离心沉淀将HGC-27细胞接种于96孔细胞培养板中，待细胞贴壁以后分别添加不同的物质(DOX-CL(45μg/mL)，NNV(40μg/mL)，SPION-NNV-DOX(40μg/mL)，SPION-NNV-DOX/MF(在外加磁场下发挥抗肿瘤作用的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂)(40μg/mL))处理HGC-27细胞；
2.按上述处理24h后，分别在96孔细胞培养板中添加CCK8检测试剂，将培养板置于CO 2孵箱中继续培养；
3.利用酶标仪检测HGC-27细胞在450nm的吸光值。
通过CCK8实验观察SPION-NNV-DOX/MF对HGC-27增殖能力的影响。结果如图5所示，通过外加磁场可以促进SPION-NNV-DOX在HGC-27细胞的积累，磁铁作用时间越长，HGC-27细胞吞噬的SPION-NNV-DOX越多；SPION-NNV-DOX/MF显著抑制HGC-27细胞增殖。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (16)

  1. 一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，其特征在于，包括以下步骤：
    1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
    2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
    3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
    4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
    5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
  2. 根据权利要求1所述制备方法，其特征在于，步骤1)每1×10 6个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。
  3. 根据权利要求1或2所述制备方法，其特征在于，步骤1)所述共同培养的温度为37℃，时间为12～24h。
  4. 根据权利要求1所述制备方法，其特征在于，步骤2)所述离心包括差速离心，所述差速离心的离心力为800g，离心时间为5min。
  5. 根据权利要求1或4所述制备方法，其特征在于，所述步骤(2)中，采用PBS进行重悬底部缓冲垫。
  6. 根据权利要求1所述制备方法，其特征在于，步骤3)所述连续物理挤压包括使用mini-extrader挤压器对所述细胞悬液进行11次连续的物理挤压。
  7. 根据权利要求1或6所述制备方法，其特征在于，步骤3)中每5mL的细胞悬液的挤压参数为：300psig，气密注射器温度为10℃以上。
  8. 根据权利要求1所述制备方法，其特征在于，步骤4)所述离心包括超速离心，所述超速离心的离心力为10000g，离心时间为80min。
  9. 根据权利要求1或8所述制备方法，其特征在于，步骤4)中，所述重悬底部NNV-药物缓冲垫用PBS进行。
  10. 根据权利要求1所述制备方法，其特征在于，步骤5)所述超顺磁材料包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。
  11. 根据权利要求1或10所述制备方法，其特征在于，步骤5)所述共孵育的温度为4℃，时间为4h。
  12. 根据权利要求1所述制备方法，其特征在于，步骤5)所述磁分离包括利用钕磁铁进行磁分离，MF＝1T。
  13. 利用权利要求1～12任一项所述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
  14. 根据权利要求13所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂，其特征在于，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂为粒径在200nm的杯状膜形囊泡。
  15. 一种体外抑制肿瘤细胞的方法，其特征在于，包括以下步骤：胰蛋白酶消化HGC-27细胞后，离心沉淀将肿瘤细胞接种于96孔细胞培养板中，待细胞贴壁以后，在外加磁场作用下，添加权利要求13或14所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂，处理24h。
  16. 根据权利要求15所述方法，其特征在于，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的添加量为40μg/mL。
PCT/CN2021/089946 2021-03-26 2021-04-26 一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法 WO2022198742A1 (zh)

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