WO2024217142A1 - 生物矿化复合结构纳米材料、制备方法及其应用 - Google Patents

生物矿化复合结构纳米材料、制备方法及其应用 Download PDF

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WO2024217142A1
WO2024217142A1 PCT/CN2024/077929 CN2024077929W WO2024217142A1 WO 2024217142 A1 WO2024217142 A1 WO 2024217142A1 CN 2024077929 W CN2024077929 W CN 2024077929W WO 2024217142 A1 WO2024217142 A1 WO 2024217142A1
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cells
derivatives
biomineralized
nanomaterial
cell
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丁显光
张晶晶
王智雨
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Nanjing University of Posts and Telecommunications
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Nanjing University of Posts and Telecommunications
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to the field of bio-material composite technology, and in particular to the preparation of biomineralized composite structures of cells and cell derivatives thereof, and their applications in new vaccines, cell derivative protection and tumor treatment.
  • Immunotherapy is to maximize the natural immune ability of the human body or help the human body restore the lost immune function. This method treats cancer by stimulating the body's own immune function, so it has no effect on other normal organs and tissues of the human body.
  • the biggest feature of immunotherapy is that it can continuously enhance the function of the human immune system.
  • experts also expect that through the auxiliary characteristics of immunotherapy, other treatment methods can achieve more ideal results.
  • Immune cell therapy refers to separating the body's immune cells (initial T cells, NK cells, etc.) from the blood, culturing them in vitro, and then reinjecting them into the body to identify and kill tumor cells.
  • Immune checkpoint blocker therapy means that the human immune system can recognize its own normal cells and not reject them. Once tumor cells learn this immune recognition method, they will not be rejected or attacked by the immune system like normal cells. By injecting immune checkpoint blockers into the body, tumor cells lose their immune recognition function and are recognized and eliminated by the immune system.
  • bio-material composite technology is a low-loss and high-efficiency treatment method: 1) It enhances the immunological response and achieves greater therapeutic benefits under low-dose conditions, which is conducive to reducing the dose and reducing potential toxic effects; 2) It can integrate multiple therapeutic functions, while integrating the biological efficacy of inner nuclear cells and their derivatives, as well as the biological activity of outer shell biometal minerals, to enhance multiple effects; 3) It protects inner nuclear cells and their derivatives, making their preservation more stable on the one hand, and avoiding the loss of activity caused by enzymatic cleavage in the body on the other.
  • the purpose of the present invention is to provide a bio-material composite technology suitable for use as a new vaccine, cell derivative protection and tumor treatment, and a biomineralized composite structure nanomaterial suitable for use as an immune checkpoint inhibitor.
  • the method of the present invention adopts mild aqueous synthesis conditions, which does not cause damage to the activity and function of the internal cells and their derivatives, so that they can maintain their original therapeutic activity; compared with traditional mineralization methods, the method of the present invention can achieve the encapsulation of internal components at a scale of tens of nanometers.
  • its biomineralized structure also has immunological activity itself, which can achieve the gain of therapeutic effect.
  • the mineralized shell layer can be subsequently chemically modified to achieve further functionalization.
  • the present invention provides a biomineralization composite structure nanomaterial with a particle size of 3 nm-20 ⁇ m, comprising:
  • the inner core is a cell and its derivatives and pathogenic microorganism structure having immune response, intercellular communication or cell proliferation functions;
  • the nanomaterials of the mineralized inner core are selected as a protective layer for cells and their derivatives, and a hydrophilic shell targeting tumor cells, wherein the shell has a modified end covalently bonded to the cells and their derivatives and a hydrophilic end targeting tumor cells.
  • the inner core is composed of cells or cell substructures or extracellular vesicle derivatives.
  • the shell is composed of metal oxides such as iron oxide, manganese oxide, aluminum oxide, etc.
  • the present invention also provides a method for preparing the above-mentioned biomineralization composite structure nanomaterial, comprising the following steps:
  • S1 dispersing cells and their derivatives having immune response, intercellular communication or cell proliferation functions in a surfactant having a concentration of less than or equal to 1 mg/mL and a pH of 7-9, and mixing them to form cells and their derivatives with surface proteins exposed;
  • step S2 adding a metal salt to the solution of cells and their derivatives with surface proteins exposed obtained in step S1, and mixing them to form metal oxide mineralized cells and their derivatives;
  • step S3 Purify the metal oxide mineralized cells and their derivatives obtained in step S2 by centrifugation.
  • the concentration of polyvinyl pyrrolidone or protein or polypeptide of the surfactant in step S1 is 1 mg/mL -10 mg/mL.
  • step S1 is 5 to 20 minutes; and the mixing time of step S2 is 30 minutes to 1 hour.
  • step S1 also includes: after mixing, centrifuging to collect cells and their derivatives with surface proteins exposed; step S2 also includes: after mixing, centrifuging to collect Fe 3+ nanoparticle precipitates.
  • the preparation method also includes the step of producing cells and their derivatives having immune response, intercellular communication or cell proliferation functions.
  • the present invention also provides the above-mentioned biomineralized composite structure nanomaterial as cells and their derivatives with immune response, intercellular communication or cell proliferation functions for application in new vaccines, cell derivative protection and tumor treatment.
  • the present invention has the following beneficial effects:
  • the nanomaterial of the present invention uses cells and their derivatives having immune response, intercellular communication or cell proliferation functions as the inner core, and mineralizes the nanomaterial of the inner core outside the inner core.
  • the selected nanomaterial serves as a protective layer for the cells and their derivatives, and a hydrophilic shell targeting tumor cells.
  • the shell has a modified end covalently bonded to the cells and their derivatives and a hydrophilic end targeting tumor cells.
  • the present invention forms a multifunctional biomineralized composite structure nanomaterial, which can be used as a new vaccine and an immune checkpoint blocker in immunotherapy. It also has tumor cell targeting, improves efficiency, and reduces toxic side effects on patients.
  • the preparation method of the biomineralized nanomaterial of the present invention has the advantages of being simple and easy to operate, easy to control the conditions, and can be produced on a large scale.
  • FIG1 is a flow chart of a method for preparing a biomineralized composite structure nanomaterial provided in an embodiment of the present invention.
  • FIG. 2 is a transmission electron microscope image of a hydrated iron oxide nanomaterial provided in an embodiment of the present invention.
  • FIG. 3 is a transmission electron microscope image of a biomineralization composite structure nanomaterial provided by an embodiment of the present invention.
  • FIG. 4 is a diagram showing the imaging treatment effect of a small animal using a biomineralized composite structure nanomaterial provided by an embodiment of the present invention.
  • the present invention utilizes nano-encapsulation technology, uses cells and their derivatives with immune response, intercellular communication or cell proliferation functions, and hydrophilic nano-materials targeting tumor cells to form biomineralized nano-structures, so as to integrate inorganic nano-materials and biological organisms.
  • the biomineralized composite structure nanomaterial of the present invention is an immunotherapy biocomposite nanomaterial, the core of which is a cell and its derivatives having immune response, intercellular communication or cell proliferation functions.
  • the cell and its derivatives having immune response, intercellular communication or cell proliferation functions of the present invention may be extracellular vesicles, proteins, HeLa cells, but other available cells and their derivatives having immunotherapy functions are not excluded.
  • the surface of the cell and its derivatives is cleaned with a surfactant to expose the surface proteins.
  • a surfactant well known to those skilled in the art, such as polyvinyl pyrrolidone, can be used.
  • commercial products can be used, and other surfactants well known to those skilled in the art can also be used.
  • the shell of the biomineralization composite structure nanomaterial is composed of hydrophilic compounds, which are used to improve the aqueous phase stability of the nanomaterial, protect the action time of the nanomaterial in the body, and can be chemically modified to provide functions such as targeting of the nanomaterial.
  • the shell has a modified end that is covalently bonded to the surface proteins of cells and their derivatives, and on the other hand, the shell has a hydrophilic end that targets tumor cells, and the hydrophilic end extends outside the nanomaterial.
  • the biocomposite structure nanomaterial of the present invention uses cells and their derivatives with immune response, intercellular communication or cell proliferation functions as the core, uses the active agent polyvinyl pyrrolidone to expose surface proteins, adsorbs trivalent iron ions on the outside of the cells and their derivatives, and uses a hydrophilic compound as its shell, forming a multifunctional biomineralization composite structure nanomaterial that can be used as a new vaccine and as an immune checkpoint blocker in immunotherapy. It also has tumor cell targeting, improves efficiency, and reduces toxic side effects on patients.
  • FIG1 shows a method flow for preparing biomineralized composite structure nanomaterials according to an embodiment of the present invention.
  • the preparation method mainly includes three steps: first, cells and derivatives thereof with immune response, intercellular communication or cell proliferation function are dispersed in a polyvinyl pyrrolidone solution of an active agent, a polyvinyl pyrrolidone substance concentration of less than or equal to 1 mg/mL, and a surfactant of pH 7-9, and mixed to form cells and derivatives with surface protein exposure.
  • the obtained surface protein-exposed cell and derivative solution thereof are dispersed in an aqueous solution of trivalent iron ions generated by oxidation of ferric permanganate, and mixed to form Fe 3+ mineralized cells and derivatives thereof. Finally, centrifugal purification is performed to obtain Fe 3+ mineralized cells and derivatives thereof. All steps of the present invention are completed at room temperature.
  • the polyvinyl pyrrolidone of the surfactant of the present invention is less than or equal to 5 mg/mL, wherein 1 mg/mL to 4 mg/mL is preferred.
  • the mixing mentioned in the preparation method can adopt conventional mixing methods, such as stirring, to make the system uniformly form a nanostructure. Depending on the selected raw materials and the relative amounts, the mixing time can generally be 5-20 minutes.
  • the present invention has no special restrictions on the source of the surfactant polyvinyl pyrrolidone, and the surfactant polyvinyl pyrrolidone well known to those skilled in the art can be used, such as commercially available products, or other surfactants well known to those skilled in the art.
  • the aqueous solution of trivalent iron ions is a hydrated iron oxide solution.
  • the mixing mentioned in the preparation method can adopt a conventional mixing method, such as stirring, to make the system uniformly form a nanostructure.
  • the mixing time is selected according to the selection of raw materials and the relative amount, which is a conventional choice made by ordinary technicians in the field.
  • the mixing time may be 30 minutes to 1 hour.
  • each nanoparticle after each nanoparticle is formed, it can be separated by centrifugation, that is, after mixing, centrifugation is performed to collect cells with surface protein exposure and their derivatives, Fe 3+ nanoparticle precipitation or biomineralization composite structure nanomaterial precipitation.
  • the present invention can also wash the obtained nanoparticles or nanomaterials.
  • the present invention has no special restrictions on the centrifugation or washing method, and the centrifugation or washing technical solutions well known to those skilled in the art can be used.
  • the washing solvent is preferably deionized water, and the number of washings is preferably 3 to 5 times.
  • the preparation method of the biomineralized structured nanomaterial of the present invention has the advantages of being simple and easy to operate, easy to control conditions, and capable of large-scale production.
  • the storage method of the biomineralized structure nanomaterial prepared by the present invention is not particularly limited. It can be dispersed in an aqueous medium and stored in a liquid form, or it can be stored in a solid form. When stored in a liquid, the aqueous medium can be one or more of double distilled water, physiological saline, a surfactant, and a tissue culture medium.
  • the particle size of the nano material obtained by the invention is 3 nm-20 ⁇ m.
  • the invention conducts transmission electron microscope analysis on the obtained trivalent iron nanomaterial.
  • the analysis result shows that the trivalent iron nanomaterial obtained by the invention aggregates into agglomerates and is amorphous-weakly crystalline hydrated iron oxide.
  • the present invention performs transmission electron microscopy observation on the biomineralization composite structure nanomaterial prepared with extracellular vesicles as the core.
  • the present invention has no special restrictions on the method of the biomineralization composite structure nanomaterial, and the technical scheme of transmission electron microscopy well known to those skilled in the art can be used.
  • the test results show that the size of the biomineralization composite structure nanomaterial of the present invention is about 20 nm-200 nm, and the structure of the extracellular vesicle can be observed without negative staining.
  • the obtained biomineralization composite structure nanomaterial can be injected into experimental animals to observe the targeting effect and immunotherapy effect of the obtained nanomaterial.
  • Example 1 of the present invention provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:
  • the present invention performs transmission electron microscopy analysis on the hydrated iron oxide nanomaterial obtained in step (2) of Example 1, and the result is shown in FIG2.
  • the hydrated iron oxide nanomaterial aggregates into a mass shape, which is amorphous-weakly crystalline hydrated iron oxide.
  • the present invention also performs transmission electron microscopy analysis on the biomineralized extracellular vesicles prepared in Example 1, and the result is shown in FIG3.
  • the biomineralized extracellular vesicles of this example are spherically distributed, with a diameter between 20 nm and 200 nm, and the structure of the extracellular vesicles can be observed without negative staining.
  • the biomineralized composite structure nanomaterial obtained in Example 1 is injected into male Balb/C tumor mice, and the distribution of the obtained biomineralized material under small animal imaging is observed. The result is shown in FIG4. As can be seen from FIG4, the injected biomineralized material is targeted to the tumor site.
  • This embodiment provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:
  • This embodiment provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:
  • This embodiment provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:
  • This embodiment provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:
  • This embodiment provides a biomineralized composite material of cells and their derivatives, which is prepared according to the following steps:

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Abstract

一种生物矿化复合结构纳米材料、制备方法及其应用，以细胞及其衍生物作为内核，矿化所述内核形成作为细胞及其衍生物的保护层以及靶向肿瘤细胞的亲水性外壳，该外壳具有与细胞及其衍生物共价键合的修饰端和靶向肿瘤细胞的亲水端。优选，内核由细胞或细胞亚结构或细胞外囊泡衍生物构成；外壳由金属盐中的铁氧化物或锰氧化物或铝氧化物构成。该纳米材料既可作为免疫治疗的新型疫苗，又可对细胞及其衍生物进行保护，还对肿瘤细胞有靶向性，提高效率，降低对患者的毒副作用；其制备方法具有简单易操作、条件易控制、可大规模生产的优点。

Description

生物矿化复合结构纳米材料、制备方法及其应用 技术领域
本发明涉及生物-材料复合技术领域，特别是涉及细胞及其细胞衍生物的生物矿化复合结构制备，以及其在新型疫苗、细胞衍生物保护以及肿瘤治疗方面的应用。
 背景技术
在中国，每天约有一万人查出患有癌症，癌症是人类健康生存的重大威胁。探索和开发各种各样的癌症治疗方法具有重要的意义。目前，癌症治疗的常见方法有“外科手术”、“化学疗法”、“放射疗法”这三种。但是这三种方法往往会给患者带来极大的痛苦和副作用。近几年，“免疫疗法”逐渐兴起，更是被称为第四种治疗方法。免疫治疗是将人体本身的自然免疫能力发挥到最大的作用，或者帮助人体恢复已经丧失的免疫功能。这种方法是通过激发人体自身的免疫功能以治疗癌症，所以对人体的其他正常器官和组织没有任何影响。免疫疗法最大的特点就是可以持续的增强人体的免疫系统功能。此外，在病发的初期，专家们也期待通过免疫疗法的辅助特性，从而使其他的治疗方式达到更理想的效果。
传统的免疫疗法包括免疫细胞治疗和免疫检查点阻滞剂治疗两种方法。免疫细胞治疗是指，把人体的免疫细胞(初始T细胞，NK细胞等)从血液里面分离出来，在体外培养后重新注入到体内，从而识别和杀死肿瘤细胞。免疫检查点阻滞剂治疗是指，人体内的免疫系统可以识别自身的正常细胞，并不予以排斥。一旦肿瘤细胞学会这种免疫识别方法后，就会像正常细胞一样不受免疫系统的排斥或者攻击。通过向体内注射免疫检查点阻滞剂，使肿瘤细胞丧失免疫识别功能，从而被免疫系统识别并消灭。
相较于传统的免疫疗法，生物-材料复合技术是一种低损高效的治疗方法：1）提升免疫学响应，实现小剂量的条件下取得更大治疗效益，有利于减少剂量减低潜在毒性作用；2）能够实现多治疗功能的集成，同时集成内核细胞及其衍生物的生物疗效，以及外壳生物金属矿化物的生物活性，多效应增强；3）保护内核细胞及其衍生物，一方面使保存更稳定，一方面能避免体内酶切导致的活性丧失。
 发明内容
本发明的目的是提供一种适合作为新型疫苗、细胞衍生物保护以及肿瘤治疗的生物-材料复合技术，同时适合作为免疫检查点抑制剂的生物矿化复合结构纳米材料。
与传统合成技术相比，由于本发明的方法采用温和水相合成条件，对内载细胞及其衍生物结构不构成活性和功能破坏，使之能保持原有治疗活性；与传统矿化方法相比，本发明的方法可实现几十纳米尺度的内载成分包裹。此外，其生物矿化结构除了能实现对内载细胞成分的活性维持，自身还具有免疫学活性，可实现治疗效应的增益。另外，该矿化外壳层可以进行后续化学修饰，实现进一步功能化。
本发明采用如下技术方案：
本发明提供了一种生物矿化复合结构纳米材料，其粒径为3 nm-20 µm，包括：
内核，所述内核为具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物及病原微生物结构；
矿化所述内核的纳米材料，所选用的纳米材料作为细胞及其衍生物的保护层，以及靶向肿瘤细胞的亲水性外壳，所述外壳具有与细胞及其衍生物共价键合的修饰端和靶向肿瘤细胞的亲水端。
进一步地，所述内核由细胞或细胞亚结构或细胞外囊泡衍生物构成。
进一步地，所述外壳由铁氧化物、锰氧化物、铝氧化物等金属氧化物构成。
本发明还提供了上述的生物矿化复合结构纳米材料的制备方法，包括如下步骤：
S1.将具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物分散于浓度小于等于1 mg/mL、pH 7-9的表面活性剂中，进行混合，以形成表面蛋白暴露的细胞及其衍生物；
S2.向步骤S1所得表面蛋白暴露的细胞及其衍生物溶液中加入金属盐，进行混合，以形成金属氧化物矿化的细胞及其衍生物；
S3.将步骤S2所得金属氧化物矿化的细胞及其衍生物进行离心纯化。
进一步地，步骤S1中表面活性剂的聚乙烯吡咯烷酮或蛋白质或多肽，浓度为1mg/mL -1 0mg/mL。
进一步地，步骤S1的混合时间为5至20分钟；步骤S2的混合时间为30分钟至1小时。
进一步地，步骤S1还包括：混合后，离心，收集表面蛋白暴露的细胞及其衍生物；步骤S2还包括：混合后，离心，收集Fe 3+纳米粒子沉淀。
进一步地，该制备方法还包括具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物的步骤。
本发明还提供了上述的生物矿化复合结构纳米材料作为具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物应用于新型疫苗、细胞衍生物保护以及肿瘤治疗等方面。
与现有技术相比，本发明的有益效果在于：
1.本发明的纳米材料以具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物作为内核，在内核外矿化所述内核的纳米材料，所选用的纳米材料作为细胞及其衍生物的保护层，以及靶向肿瘤细胞的亲水性外壳，所述外壳具有与细胞及其衍生物共价键合的修饰端和靶向肿瘤细胞的亲水端。
2.本发明形成了一种具有多功能的生物矿化复合结构纳米材料，既可作为新型疫苗，又可作为免疫治疗中的免疫检查点阻滞剂，还具有肿瘤细胞靶向性，提高效率，降低对患者的毒副作用。
3.本发明的生物矿化纳米材料的制备方法具有简单易操作、条件易控制、可大规模生产的优点。
 附图说明
图1是本发明实施例提供的一种生物矿化复合结构纳米材料的制备方法的流程图。
图2是本发明实施例提供的一种水合氧化铁纳米材料的透射电镜图。
图3是本发明实施例提供的一种生物矿化复合结构纳米材料的透射电镜图。
图4是本发明实施例提供的一种生物矿化复合结构纳米材料的小动物成像治疗效果图。
 具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明利用了纳米包裹技术，使用具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物、靶向肿瘤细胞的亲水性纳米材料形成生物矿化纳米结构，以将无机纳米材料和生物有机体集为一体。
本发明的生物矿化复合结构纳米材料为免疫治疗生物复合纳米材料，其内核为具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物。例如，本发明的具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物可为细胞外囊泡、蛋白质、海拉细胞，但不排除其他可用的具有免疫治疗功能的细胞及其衍生物。
矿化所述内核之前采用表面活性剂对细胞及其衍生物表面进行清洗，暴露表面蛋白。本发明对表面活性剂的来源没有特殊的限制，采用本领域技术人员熟知的表面活性剂如聚乙烯吡咯烷酮即可，如可以采用市售商品，也可以采用本领域技术人员熟知的其他表面活性剂。
生物矿化复合结构纳米材料的外壳由亲水性化合物构成，用于提高纳米材料的水相稳定性，保护纳米材料在体内的作用时间，并可以进行化学修饰以提供纳米材料的靶向性等功能，一方面，该外壳具有与细胞及其衍生物表面蛋白共价键合的修饰端，另一方面，该外壳具有靶向肿瘤细胞的亲水端，该亲水端延伸在该纳米材料的外部。
本发明的生物复合结构纳米材料以具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物作为内核，采用活性剂聚乙烯吡咯烷酮暴露表面蛋白，在细胞及其衍生物外吸附有三价铁离子，并将亲水性化合物作为其外壳，形成了一种具有多功能的生物矿化复合结构纳米材料，即可作为新型疫苗，又可作为免疫治疗中的免疫检查点阻滞剂，还具有肿瘤细胞靶向性，提高效率，降低对患者的毒副作用。
图1示出了根据本发明的实施方案，制备生物矿化复合结构纳米材料的方法流程。从图1所示可见，制备方法主要包括三个步骤：首先，将免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物分散于活性剂聚乙烯吡咯烷酮溶液中，聚乙烯吡咯烷酮物质浓度小于等于1 mg/mL、pH 7-9的表面活性剂中，进行混合，以形成表面蛋白暴露的细胞及其衍生物。之后，将所得表面蛋白暴露的细胞及其衍生物溶液分散于高锰酸铁氧化生成的三价铁离子的水溶液中，进行混合，以形成Fe 3+矿化的细胞及其衍生物。最后，离心纯化，获取Fe 3+矿化的细胞及其衍生物。本发明的所有步骤，均在室温下完成。
在活性剂聚乙烯吡咯烷酮暴露细胞及其衍生物表面蛋白步骤中，为了保证聚乙烯吡咯烷酮暴露内核外蛋白的效果及反应效率，本发明的表面活性剂的聚乙烯吡咯烷酮小于等于5 mg/mL，其中，以1 mg/mL 至4 mg/mL为佳。制备方法中所提及的混合，可以采用常规的混合方法，如搅拌，来使体系均匀地形成纳米结构。根据选用原料以及相对量的不同，混合时间通常可以为5-20分钟。本发明对表面活性剂聚乙烯吡咯烷酮的来源没有特殊的限制，采用本领域技术人员熟知的表面活性剂聚乙烯吡咯烷酮即可，如可以采用市售商品，也可以采用本领域技术人员熟知的其他表面活性剂。
之后，在Fe 3+的吸附步骤中，三价铁离子的水溶液为水合氧化铁溶液。制备方法中所提及的混合，可以采用常规的混合方法，如搅拌，来使体系均匀地形成纳米结构。混合时间是根据选用原料以及相对量的不同进行选择的，属于本领域普通技术人员作出的常规选择。
最后，在纳米材料矿化生物有机体形成步骤中，混合时间可为30分钟至1小时。
上述的三个步骤中在各个纳米粒子形成之后，可以采用离心的方式进行分离，即混合后，离心，收集表面蛋白暴露的细胞及其衍生物、Fe 3+纳米粒子沉淀或生物矿化复合结构纳米材料沉淀。本发明的还可为所得上述各个纳米粒子或纳米材料进行洗涤，本发明对所述的离心或洗涤的方法没有特殊的限制，采用本领域技术人员熟知的离心或洗涤的技术方案即可。在本发明中，所述洗涤的溶剂优选为去离子水，所述洗涤的次数优选为3次到5次。
本发明的生物矿化结构纳米材料的制备方法具有简单易操作、条件易控制、可大规模生产的优点。
本发明所制备的生物矿化结构纳米材料的保存方式没有特殊的限制，可以将其分散在水性介质中，以液态形式保存，也可以以固体形式保存。以液体保存时，水性介质可为双蒸水、生理盐水、表面活性剂、组织培养液中的一种或多种。
本发明所得的纳米材料的粒径为3 nm-20 µm。
本发明对所得的三价铁纳米材料进行了透射电镜分析。分析结果表明：本发明所得的三价铁纳米聚集成团块状，为无定形-弱结晶的水合氧化铁。
本发明对以细胞外囊泡为内核制备的生物矿化复合结构纳米材料进行透射电子显微镜观察。本发明对所述生物矿化复合结构纳米材料的方法没有特殊的限制，采用本领域技术人员熟知的透射电子显微镜的技术方案即可。测试结果表明：本发明的生物矿化复合结构纳米材料尺寸在20 nm-200 nm左右，无需负染也能观察到细胞外囊泡的结构。
在本发明的实施例中，可以将所得生物矿化复合结构纳米材料注入实验动物体内，观察所得纳米材料的靶向效果以及免疫治疗效果。
实施例1：
本发明实施例1提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）通过梯度超速离心获取细胞外囊泡，用1 ml PBS重悬细胞外囊泡。
（2）1 M高锰酸铁在水中反应生成水合氧化铁。
（3）取100 μl所制取得细胞外囊泡悬浊液分散于1 ml PBS溶液中，加入20μl水合氧化铁纳米材料，搅拌5 min，离心获取水合氧化铁矿化的细胞外囊泡。
本发明对实施例1步骤（2）所得水合氧化铁纳米材料进行了透射电镜分析，结果如图2所示，由图2可知，水合氧化铁纳米材料聚集成团块状，为无定形-弱结晶的水合氧化铁。本发明还对实施例1所制备的生物矿化细胞外囊泡进行了透射电镜分析，结果如图3所示，由图3可知，本实施例的生物矿化外囊泡呈球形分布，直径在20 nm-200 nm之间，无需负染也能观察到细胞外囊泡的结构。将实施例1所得生物矿化复合结构纳米材料注入雄性Balb/C肿瘤鼠体内，观察所得生物矿化材料在小动物成像下的分布情况，结果如图4所示，由图4可知，注入的生物矿化材料靶向到肿瘤部位。
实施例2：
本实施例提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）胰酶消化1x106个海拉细胞，用1 ml PBS重悬消化后的细胞；
（2）1 M高锰酸铁在水中反应生成水合氧化铁；
（3）取100 μl所制取得海拉细胞悬浊液分散于1 ml PBS溶液中，加入20μl水合氧化铁纳米材料，搅拌5 min，离心获取水合氧化铁矿化的海拉细胞。
实施例3：
本实施例提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）收集蛋白质；
（2）1 M高锰酸铁在水中反应生成水合氧化铁；
（3）取100 μl所制取得蛋白质悬浊液分散于1 ml PBS溶液中，加入20μl水合氧化铁纳米材料，搅拌5 min，离心获取水合氧化铁矿化的蛋白质。
实施例4：
本实施例提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）胰酶消化1x106个海拉细胞，用1 ml PBS重悬消化后的细胞；
（2）1 M高锰酸钾在水中被还原为二氧化锰；
（3）取100 μl所制取得海拉细胞悬浊液分散于1 ml PBS溶液中，加入20μl二氧化锰溶液，搅拌5 min，离心获取二氧化锰矿化的海拉细胞。
实施例5：
本实施例提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）通过梯度超速离心获取细胞外囊泡，用1 ml PBS重悬细胞外囊泡；
（2）制备氢氧化铜；
（3）取100 μl所制取得海拉细胞悬浊液分散于1 ml PBS溶液中，加入20μl氢氧化铜纳米材料，搅拌5 min，离心获取氢氧化铜矿化的海拉细胞。
实施例6：
本实施例提供了一种细胞及其衍生物生物矿化复合材料，按照如下步骤制备：
（1）胰酶消化1x10 6个海拉细胞，用1 ml PBS重悬消化后的细胞；
（2）制备二氧化镁；
（3）取100 μl所制取得海拉细胞悬浊液分散于1 ml PBS溶液中，加入20μl二氧化镁，搅拌5 min，离心获取二氧化镁矿化的海拉细胞。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 一种生物矿化复合结构纳米材料，其特征在于，其粒径为3 nm-20 µm，包括：
    内核，所述内核为具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物；
    矿化所述内核的纳米材料，所选用的纳米材料作为细胞及其衍生物的保护层以及靶向肿瘤细胞的亲水性外壳，所述外壳具有与细胞及其衍生物共价键合的修饰端和靶向肿瘤细胞的亲水端。
  2. 根据权利要求1所述的生物矿化复合结构纳米材料，其特征在于，所述内核由细胞或细胞亚结构或细胞外囊泡衍生物构成；
    所述外壳由金属盐中的铁氧化物或锰氧化物或铝氧化物构成。
  3. 一种制备权利要求2所述的生物矿化复合结构纳米材料的方法，其特征在于，包括如下步骤：
    S1.将具有免疫应答、细胞间通讯或细胞增殖功能的细胞及其衍生物分散于浓度小于等于1 mg/mL、pH 7-9的表面活性剂中，进行混合，以形成表面蛋白暴露的细胞及其衍生物；
    S2.向步骤S1所得表面蛋白暴露的细胞及其衍生物溶液中加入金属盐，进行混合，以形成金属氧化物矿化的细胞及其衍生物；
    S3.将步骤S2所得金属氧化物矿化的细胞及其衍生物进行离心纯化。
  4. 根据权利要求3所述的生物矿化复合结构纳米材料的制备方法，其特征在于，步骤S1中的表面活性剂采用聚乙烯吡咯烷酮或蛋白质或多肽，其浓度为1mg/mL -10 mg/mL。
  5. 根据权利要求3所述的生物矿化复合结构纳米材料的制备方法，其特征在于，步骤S1的混合时间为5-20分钟；步骤S3的混合时间为30-60分钟。
  6. 根据权利要求3所述的生物矿化复合结构纳米材料的制备方法，其特征在于，步骤S1还包括：混合后，离心，收集表面蛋白暴露的细胞及其衍生物；
    步骤S2还包括：混合后，离心，收集Fe 3+纳米粒子沉淀。
  7. 权利要求1所述的生物矿化复合结构纳米材料的应用，其特征在于，其能够应用于新型疫苗或细胞衍生物保护。
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