WO2021077770A1 - 一种迷你联合佐剂纳米颗粒及其制备方法和应用 - Google Patents

一种迷你联合佐剂纳米颗粒及其制备方法和应用 Download PDF

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WO2021077770A1
WO2021077770A1 PCT/CN2020/096576 CN2020096576W WO2021077770A1 WO 2021077770 A1 WO2021077770 A1 WO 2021077770A1 CN 2020096576 W CN2020096576 W CN 2020096576W WO 2021077770 A1 WO2021077770 A1 WO 2021077770A1
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adjuvant
nanoparticle
combined
combined adjuvant
nanoparticles
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PCT/CN2020/096576
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刘兰霞
刘丹
刘佳乐
冷希岗
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中国医学科学院生物医学工程研究所
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Priority to US17/256,095 priority patent/US20220241407A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A

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  • the invention belongs to the field of biotechnology, and specifically relates to a joint adjuvant nanoparticle prepared by self-assembly, and a preparation method and application thereof.
  • Adjuvants are injected into animals prior to antigens or at the same time, which can non-specifically change the body's specific immune response to antigens, enhance the immunogenicity of the corresponding antigens or change the type of immune response, but have no antigenic properties themselves.
  • the main role of adjuvants is to present antigens and enhance the stimulation of the immune system.
  • Immune enhancers directly (such as cytokines) or through pattern recognition receptors (PRR) (such as bacterial components) activate innate immunity.
  • the commonly used adjuvants in my country include aluminum salt, oil emulsion, propolis, polysaccharides, microbial Freund's (FA) adjuvant, ⁇ -interferon (IFN- ⁇ ), interleukin (Interleuki-ns, ILs), immune stimulation Complexes (ISCOMs), glycosides and compound traditional Chinese medicine adjuvants
  • new immune adjuvants include nucleic acid, CpG, complement, nano, liposome (LIP) or the combined application of two or more adjuvants.
  • newly-marketed vaccines often need to be co-delivered with the aid of a delivery system loaded with antigen and adjuvant to induce the body to produce a stronger protective immune response.
  • commonly used vaccine delivery systems are generally made of biomedical materials. However, there are potential safety problems after biomedical materials enter the human body.
  • the present invention provides a combination adjuvant nanoparticle.
  • the present invention provides a combined adjuvant nanoparticle, the combined adjuvant nanoparticle is prepared by self-assembly of amphiphilic monomer molecules, and the amphiphilic monomer molecule is a hydrophobic adjuvant The molecule reacts with the hydrophilic adjuvant molecule.
  • the diameter of the combined adjuvant nanoparticles is 100-200 nm.
  • the hydrophobic adjuvant molecule is monophosphatidyl lipid A or an analog thereof.
  • the hydrophilic adjuvant molecule is an oligonucleotide or oligodeoxynucleotide.
  • the hydrophilic adjuvant molecule is CPG-ODN.
  • the present invention provides a method for preparing combined adjuvant nanoparticles, which includes the following steps:
  • S2 Mix the material obtained from S1 with oligodeoxynucleotides, stir at room temperature for 12-18 hours, then dialyze and freeze-dry to obtain the combined adjuvant nanoparticles.
  • adding diphenyl azide phosphate and 1,8-diazabicyclo[5.4.0]undec-7-ene to modify the azide group, stirring at 20°C for 24 to 48 hours Dialysis and lyophilization; the ratio of the mass of the monophosphatidyl lipid A to the volume of diphenyl azide phosphate and the volume of 1,8-diazabicyclo[5.4.0]undec-7-ene ( 2.0 ⁇ 4.0)mg: (3.0 ⁇ 6.0) ⁇ l: (2.0 ⁇ 4.0) ⁇ l.
  • step S2 the mass ratio of the substance obtained in S1 to the volume of oligodeoxynucleotides is (1.0-2.0) mg: (100-200) ⁇ l.
  • dialysis bags are used for dialysis, and substances with larger molecular weights are collected during dialysis.
  • the loaded material is selected from drugs and antigens.
  • it is chicken ovalbumin.
  • it can also be other antigens or drugs
  • the present invention provides the application of the aforementioned combined adjuvant nanoparticles in the preparation of drug and antigen-loaded complexes.
  • the present invention provides an immunogenic composition containing an effective amount of antigen and the aforementioned combined adjuvant nanoparticles.
  • the material obtained by S2 is mixed with the material to be loaded, and the reaction is stirred at room temperature for 8-10 hours to obtain nanoparticles of the immunogenic composition.
  • the load to be loaded can be chicken ovalbumin or other antigens or drugs.
  • the mass ratio of the material obtained by the S2 to the material to be loaded is 1:1-2.
  • the present invention also provides the application of the above-mentioned composition in the preparation of a vaccine for the treatment or prevention of tumors or tuberculosis.
  • the combined adjuvant nanoparticle of the present invention uses the amphiphilic monomer molecule formed by the hydrophilic adjuvant molecule and the hydrophobic adjuvant molecule as a primitive, and is prepared by self-assembly; the combined adjuvant nanoparticle has a relatively stimulating effect
  • the combined application of hydrophilic adjuvant and hydrophobic adjuvant in the free state has a stronger effect.
  • the combined adjuvant nanoparticles can also be used as nanocarriers to deliver antigens to antigen-presenting cells, promote the uptake of antigens by antigen-presenting cells, realize the co-delivery of antigen and adjuvant, produce a synergistic immune response, and can greatly enhance anti-tumor Immune efficacy of vaccines such as TB and tuberculosis.
  • Figure 1 is a diagram showing the results of the combined adjuvant nanoparticle dynamic light scattering particle size detection
  • Figure 2 is a transmission electron microscope image of combined adjuvant nanoparticles
  • Figure 3 is the cell viability detection diagram after nanoparticle acts on DC
  • Figure 4 is the result of confocal imaging of the uptake of nanoparticles by DC cells
  • Figure 5 is a diagram showing the effect of nanoparticles on the promotion of DC cell maturation
  • Figure 6 is a diagram showing the secretion of cytokines after DC cells are incubated with nanoparticles.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to three repeated experiments, and the data is the average value of the three repeated experiments or the average ⁇ standard deviation.
  • the present invention provides a combined adjuvant nanoparticle, which is prepared by self-assembly by using an amphiphilic monomer molecule as a self-assembly element, and the amphiphilic monomer molecule is a hydrophobic adjuvant molecule and a hydrophilic adjuvant molecule The response is obtained.
  • the hydrophilic adjuvant molecule is an oligodeoxynucleotide CPG-ODN.
  • the hydrophobic adjuvant molecule is monophosphatidyl lipid A (MPLA) or its analogues.
  • the hydrophilic adjuvant molecule is an oligodeoxyribonucleotide CPG-ODN.
  • CPG-ODN oligodeoxyribonucleotide
  • it is Type C 2395
  • sequence of CPG-ODN is: 5'-TCGTCGTTTTCGGCGCGCGCCG-3', purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the hydrophobic adjuvant molecule reacts with diphenyl azide phosphate (DPPA) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to The hydrophobic adjuvant molecule is modified with an azide group.
  • DPPA diphenyl azide phosphate
  • DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
  • the mass ratio of monophosphatidyl lipid A (MPLA) to the volume of DPPA and DBU is: (2.0 ⁇ 4.0) mg: (3.0 ⁇ 6.0) ⁇ l: (2.0 ⁇ 4.0) ⁇ l.
  • the mass ratio of monophosphatidyl lipid A azide substance to the volume of CPG-ODN is (1.0-2.0) mg: (100-200) ⁇ l.
  • the specific steps of preparing the combined adjuvant nanoparticles include:
  • FIG. 1 is a diagram showing the results of the dynamic light scattering particle size detection of the combined adjuvant nanoparticles prepared in Example 1
  • FIG. 2 is a transmission electron microscope diagram of the combined adjuvant nanoparticles.
  • the resulting combined adjuvant nanoparticles have a diameter of 136.9-138.6 nm.
  • the dispersion index of the combined adjuvant nanoparticles is 0.11-0.16.
  • MPLA-CPG nanoparticles 1.0 mg MPLA-CPG nanoparticles and 1.0 mg chicken ovalbumin (OVA) were stirred and reacted at room temperature for 10 hours to obtain MPLA-CPG-OVA nanoparticles.
  • OVA ovalbumin
  • C57BL/6 mouse bone marrow-derived dendritic cells (Bone Marrow Derived Dendritic Cell, BMDC) and place them in a 5% CO 2 incubator at 37°C for culture. On the seventh day, gently pipette the culture medium , Collect suspended cells and loose adherent growth cells, inoculate them in 96-well plates, overnight in an incubator, and add concentrations of 0, 1, 5, 10, 20, 30 ⁇ g/ml to each well to load OVA (chicken ovalbumin).
  • OVA thick ovalbumin
  • MPLA-CPG nanoparticles continue to incubate for 24 hours, add 10 ⁇ l of CCK-8 detection solution to each well, continue to incubate for 1 to 4 hours in an incubator, and measure the absorbance at 450 nm with a multifunctional full-wavelength microplate reader (ThermoVarioskan Flash3001).
  • a multifunctional full-wavelength microplate reader ThermoVarioskan Flash3001.
  • the results obtained after the nanoparticles act on DC cells are shown in Figure 3.
  • BMDCs In order to observe the uptake of BMDCs to nanoparticles, OVA was labeled with FITC, the cells were plated in a confocal dish, and BMDCs were incubated with FITC-OVA-loaded MPLA-CPG nanoparticles for 6 hours (calculated according to the OVA concentration 10 ⁇ g/ml Dosage), washed with PBS, fixed with fixative, stained lysosomes with Lyso-Tracker Red, stained nuclei with DAPI, and observed MPLA-CPG nanoparticles loaded with FITC-OVA using a confocal laser microscope (Leica, TCS SP5) Distributed in BMDCs, the whole process is protected from light.
  • FITC-OVA-loaded MPLA-CPG nanoparticles for 6 hours (calculated according to the OVA concentration 10 ⁇ g/ml Dosage), washed with PBS, fixed with fixative, stained lysosomes with Lyso-Tracker
  • Flow cytometry was used to detect the effects of nanoparticles on the promotion of BMDCs cell maturation.
  • BMDC and MPLA-CPG nanoparticles loaded with OVA were incubated for 8 hours (calculated according to the OVA concentration of 10 ⁇ g/ml), and the cells were collected and labeled with CD11C and CD40.
  • CD80 and other flow cytometry antibodies use flow cytometry to detect.
  • Use PBS the same concentration of free OVA and a mixture of OVA+MPLA+CPG as a control.
  • BMDCs were collected and seeded in 96-well plates. After BMDC and MPLA-CPG nanoparticles loaded with OVA were incubated for 8 hours (calculated according to the OVA concentration of 10 ⁇ g/ml to calculate the dose), centrifuged to discard the supernatant medium, replaced with a new medium, and continued to culture for 24 hours, according to the ELISA kit instructions Methods The cytokine IFN- ⁇ (Interferon- ⁇ , Interferon- ⁇ ) and TNF- ⁇ (Tumor Necrosis Factor- ⁇ , Tumor Necrosis Factor- ⁇ ) were determined in the supernatant of BMDCs.
  • IFN- ⁇ Interferon- ⁇ , Interferon- ⁇
  • TNF- ⁇ Tumor Necrosis Factor- ⁇

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Abstract

本发明公开了一种以两亲性单体分子为基元通过自组装制备而成的联合佐剂纳米颗粒，所述两亲性单体分子为疏水性佐剂分子和亲水性佐剂分子反应得到。

Description

一种迷你联合佐剂纳米颗粒及其制备方法和应用
本申请要求于2019年10月23日提交中国专利局、申请号为201911012900.7、发明名称为“一种迷你联合佐剂纳米颗粒及其制备方法和应用”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
本发明属于生物技术领域，具体涉及一种自组装制备而成的联合佐剂纳米颗粒及其制备方法和应用。
背景技术
佐剂是先于抗原或同时注射于动物体内，能非特异性改变机体对抗原的特异性免疫应答，能增强相应抗原的免疫原性或改变免疫反应类型，而本身并无抗原性的物质。佐剂的主要作用是递呈抗原和增强免疫系统的刺激。免疫增强剂直接(如细胞因子)或通过模式识别受体(pattern recognition receptor，PRR)(如细菌组分)激活固有免疫。目前，我国常用的佐剂有铝盐、油乳、蜂胶、多糖、微生物氟氏(FA)佐剂、γ-干扰素(IFN-γ)、白细胞介素(Interleuki-ns，ILs)、免疫刺激复合物(ISCOMs)、糖苷及复方中药佐剂等，新型免疫佐剂有核酸、CpG、补体、纳米、脂质体(LIP)或两个以上佐剂联合应用等。为了增强疫苗效果，新上市的疫苗常需要借助递送系统同时负载抗原和佐剂进行共递送，诱导机体产生更强的保护性免疫反应。目前，常用的疫苗递送系统一般是由生物医用材料制备而成，然而，生物医用材料进入人体后存在潜在的安全性问题。
发明内容
针对现有技术中的缺陷，本发明提供一种联合佐剂纳米颗粒。
在一个方面，本发明提供了一种联合佐剂纳米颗粒，所述联合佐剂纳米颗粒为两亲性单体分子通过自组装制备而成，所述两亲性单体分子为疏水性佐剂分子和亲水性佐剂分子反应得到。
在一些实施方案中，所述联合佐剂纳米颗粒的直径为100-200nm。
在一些实施方案中，所述疏水性佐剂分子为单磷脂酰脂质A或其类似物。
在一些实施方案中，所述亲水性佐剂分子为寡聚核苷酸或寡聚脱氧核苷酸。优选地，所述亲水性佐剂分子为CPG-ODN。
在另一个方面，本发明提供了一种联合佐剂纳米颗粒的制备方法，包括如下步骤：
S1：将单磷脂酰脂质A或其类似物进行叠氮化基团修饰，透析、冻干；
S2：将S1所得物质与寡聚脱氧核苷酸混合，室温下搅拌12-18h，然后透析、冻干，得到所述联合佐剂纳米颗粒。
在一些实施方案中，加入叠氮磷酸二苯酯和1,8-二氮杂双环[5.4.0]十一碳-7-烯进行叠氮化基团修饰，20℃搅拌反应24～48h后透析、冻干；所述单磷脂酰脂质A的质量与叠氮磷酸二苯酯的体积、1,8-二氮杂双环[5.4.0]十一碳-7-烯的体积的比值(2.0～4.0)mg：(3.0～6.0)μl：(2.0～4.0)μl。
在一些实施方案中，步骤S2中，S1所得物质的质量与寡聚脱氧核苷酸的体积比为(1.0～2.0)mg：(100～200)μl。
具体地，采用透析袋进行透析，透析时收集分子量较大的物质。
在一些实施方案中，所述带负载物选自药物、抗原。优选为鸡卵清蛋白。当然也可以为其他抗原或药物
在又一个方面，本发明提供了上述联合佐剂纳米颗粒在制备负载药物、抗原的复合物中的应用。
在又一个方面，本发明提供了一种免疫原性组合物，其含有有效量抗原和上述的联合佐剂纳米颗粒。
在一些实施方案中，将S2得到的物质与待负载物混合，室温下搅拌反应8-10h，得到免疫原性组合物纳米颗粒。所述待负载物可以为鸡卵清蛋白或其它抗原或药物。当所述待负载物为鸡卵清蛋白时，所述S2得到的物质与所述待负载物的质量比为1：1-2。
本发明还提供了上述组合物在制备治疗或预防肿瘤或结核的疫苗中的应用。
本发明的有益效果体现在：
本发明的联合佐剂纳米颗粒以亲水性佐剂分子和疏水性佐剂分子形 成的两亲性单体分子作为基元，通过自组装制备而成；该联合佐剂纳米颗粒的刺激作用相对于游离状态下联合应用亲水性佐剂和疏水性佐剂产生的作用更强。而且，联合佐剂纳米颗粒还可作为纳米载体递送抗原至抗原提呈细胞，促进抗原提呈细胞对抗原的摄取，实现抗原与佐剂的共递送，产生协同性免疫应答，能够大大增强抗肿瘤、结核等疫苗的免疫疗效。
附图说明
图1为联合佐剂纳米颗粒动态光散射粒径检测结果图；
图2为联合佐剂纳米颗粒透射电子显微镜图；
图3为纳米颗粒作用于DC后的细胞活力检测图；
图4为DC细胞摄取纳米颗粒共聚焦成像结果图；
图5为纳米颗粒对DC细胞的促成熟情况图；
图6为DC细胞与纳米颗粒孵育后细胞因子的分泌情况图。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只作为示例，而不能以此来限制本发明的保护范围。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
下面的实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到。以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。
本发明提供一种联合佐剂纳米颗粒，其采用两亲性单体分子作为自组装基元通过自组装制备而成，该两亲性单体分子为疏水佐剂分子和亲水性佐剂分子反应得到。
作为本发明的一个实施例，亲水性佐剂分子为寡聚脱氧核苷酸CPG-ODN。
作为本发明的一个实施例，疏水性佐剂分子为单磷脂酰脂质A(MPLA)或其类似物。
作为本发明的一个实施例，亲水性佐剂分子为寡聚脱氧核糖核苷酸CPG-ODN。优选为C型2395，CPG-ODN的序列(SEQ ID NO.1)为：5’-TCGTCGTTTTCGGCGCGCGCCG-3’，购自生工生物工程(上海)股份有限公司。
作为本发明的一个实施例，疏水性佐剂分子与叠氮磷酸二苯酯(DPPA)和1,8-二氮杂双环[5.4.0]十一碳-7-烯(DBU)反应以对疏水性佐剂分子进行叠氮化基团修饰。
疏水性佐剂分子为单磷脂酰脂质A时，单磷脂酰脂质A(MPLA)的质量与DPPA、DBU的体积比为：(2.0～4.0)mg：(3.0～6.0)μl：(2.0～4.0)μl。单磷脂酰脂质A叠氮化的物质的质量与CPG-ODN的体积比为(1.0～2.0)mg：(100～200)μl。
实施例1
如下式所示，为本发明一个实施例的单磷脂酰脂质A(MPLA)与CPG-ODN(序列为SEQID NO.1)的合成结构路线：
Figure PCTCN2020096576-appb-000001
其中，
Figure PCTCN2020096576-appb-000002
联合佐剂纳米颗粒制备的具体步骤包括：
S1：将4.0mg单磷脂酰脂质A(MPLA)、6.0μl叠氮磷酸二苯酯(DPPA)和4.0μl 1,8-二氮杂双环[5.4.0]十一碳-7-烯(DBU)混合，20℃下搅拌反应48h，对所得产物用透析袋进行透析，收集分子量大于1000kD的物质，冻干；
S2：将2.0mg冻干后的物质与200μl功能性寡聚脱氧核苷酸CPG-ODN混合，室温下搅拌过夜，然后透析袋进行透析，收集分子量大于2000kD的物质，冻干，得到MPLA-CPG联合佐剂纳米颗粒。
图1为实施例1制备得到的联合佐剂纳米颗粒动态光散射粒径检测结果图，图2为该联合佐剂纳米颗粒透射电子显微镜图。
得到的联合佐剂纳米颗粒的直径为136.9-138.6nm。联合佐剂纳米颗粒的分散度指数为0.11-0.16。
实施例2
将1.0mg MPLA-CPG纳米颗粒与1.0mg鸡卵清蛋白(OVA)，室温下搅拌反应10h，得到MPLA-CPG-OVA纳米颗粒。
实施例3
1、细胞活力实验
取6～8周龄的C57BL/6小鼠骨髓来源树突状细胞(Bone Marrow DerivedDendritic Cell，BMDC)，置于5％CO 2的37℃培养箱中进行培养，第七天，轻柔吹打培养液，收集悬浮细胞及疏松贴壁生长的细胞，接种于96孔板中，培养箱中过夜，每孔加入浓度分别为0、1、5、10、20、30μg/ml负载OVA(鸡卵清蛋白)的MPLA-CPG纳米颗粒，继续培养24小时，每孔加入10μl CCK-8检测液，培养箱中继续培养1～4h，利用多功能全波长酶标仪(ThermoVarioskanFlash3001)测定450nm处吸光度。用同浓度的游离OVA和OVA+MPLA+CPG三者的混合物作对照。纳米颗粒作用于DC细胞后所得结果如图3所示。
2、细胞摄取实验
为了观察BMDCs对于纳米颗粒的摄取情况，OVA用FITC进行标记，将细胞铺于共聚焦皿中，BMDCs与负载FITC-OVA的MPLA-CPG纳米颗粒共孵育6h后(按照OVA的浓度10μg/ml计算给药量)，PBS进行洗涤，固定液进行固定，用Lyso-Tracker Red染溶酶体，DAPI染细胞核，利用激光共聚焦显微镜(Leica，TCS SP5)观察负载FITC-OVA的MPLA-CPG纳米颗粒在BMDCs中分布状况，全程避光操作。用同浓度的游离FITC-OVA作对照，DC细胞摄取纳米颗粒共聚焦成像结果如图4所示。结果显示，BMDCs对MPLA-CPG-OVA纳米颗粒的摄入量显著。
3、纳米颗粒对BMDCs成熟的影响
采用流式细胞术检测纳米颗粒对于BMDCs细胞促成熟情况，BMDC与负载OVA的MPLA-CPG纳米颗粒共孵育8小时(按照OVA的浓度 10μg/ml计算给药量)，收集细胞，标记CD11C、CD40及CD80等流式抗体，用流式细胞仪进行检测。用PBS、同浓度的游离OVA和OVA+MPLA+CPG三者的混合物作对照。纳米颗粒对DC细胞的促成熟结果如图5所示，其中Free OVA用Free O表示，Free MPLA+CPG+OVA用Free MCO表示，MPLA-CPG-OVANPs用MCO NPs表示。
采用ELISA法测定纳米颗粒对BMDCs细胞因子分泌的影响：收集BMDCs，接种于96孔板中。BMDC与负载OVA的MPLA-CPG纳米颗粒共孵育8小时(按照OVA的浓度10μg/ml计算给药量)后，离心弃上清培养基，更换新培养基，继续培养24h，按照ELISA试剂盒说明书方法对BMDCs上清液进行细胞因子IFN-γ(Interferon-γ，干扰素-γ)和TNF-α(TumorNecrosisFactor-α，肿瘤坏死因子-α)的含量测定。使用酶标仪测定450nm处的吸光度OD值，根据标准品吸光度与浓度绘制标准曲线，计算样品浓度。用PBS、同浓度的游离OVA和OVA+MPLA+CPG三者的混合物作对照，DC细胞与纳米颗粒孵育后细胞因子的分泌情况如图6所示。结果显示，MPLA-CPG-OVA纳米颗粒可显著促进BMDCs细胞因子分泌，具有促进抗原提呈细胞活化的作用。
从图3至图6的结果可知，该纳米颗粒的亲水性佐剂分子和疏水性佐剂分子游离状态下联用也具有协同刺激作用，但将二者连接制备成联合佐剂纳米颗粒后，比游离状态下联合应用产生的协同作用的程度大大增强。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。

Claims (10)

  1. 一种联合佐剂纳米颗粒，其特征在于，所述联合佐剂纳米颗粒为两亲性单体分子通过自组装制备而成，所述两亲性单体分子为疏水性佐剂分子和亲水性佐剂分子反应得到。
  2. 根据权利要求1所述的联合佐剂纳米颗粒，其特征在于，所述联合佐剂纳米颗粒的直径为100-200nm。
  3. 根据权利要求1所述的联合佐剂纳米颗粒，其特征在于，所述疏水性佐剂分子为单磷脂酰脂质A或其类似物。
  4. 根据权利要求1所述的联合佐剂纳米颗粒，其特征在于，所述亲水性佐剂分子为寡聚核苷酸或寡聚脱氧核苷酸。
  5. 根据权利要求4所述的联合佐剂纳米颗粒，其特征在于，所述亲水性佐剂分子为CPG-ODN。
  6. 一种联合佐剂纳米颗粒的制备方法，其特征在于，包括如下步骤：
    S1：将单磷脂酰脂质A或其类似物进行叠氮化基团修饰，透析、冻干；
    S2：将S1所得物质与寡聚脱氧核苷酸混合，室温下搅拌12-18h，然后透析、冻干，得到所述联合佐剂纳米颗粒。
  7. 根据权利要求6所述的制备方法，其特征在于，步骤S1中，加入叠氮磷酸二苯酯和1,8-二氮杂双环[5.4.0]十一碳-7-烯进行叠氮化基团修饰，20℃搅拌反应24～48h后透析、冻干；所述单磷脂酰脂质A的质量与叠氮磷酸二苯酯的体积、1,8-二氮杂双环[5.4.0]十一碳-7-烯的体积的比值(2.0～4.0)mg：(3.0～6.0)μl：(2.0～4.0)μl；步骤S2中，S1所得物质的质量与寡聚脱氧核苷酸的体积比为(1.0～2.0)mg：(100～200)μl。
  8. 权利要求1-5任意一项所述的联合佐剂纳米颗粒在制备负载药物、抗原的复合物中的应用。
  9. 一种免疫原性组合物，其特征在于，含有有效量抗原和权利要求1-5任意一项所述的联合佐剂纳米颗粒。
  10. 权利要求9所述的免疫原性组合物在制备治疗或预防肿瘤或结核的疫苗中的应用。
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CN114796476A (zh) * 2021-09-24 2022-07-29 中国医学科学院医学生物学研究所 一种亚单位疫苗新型核酸佐剂系统及其应用
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015192603A1 (zh) * 2014-06-18 2015-12-23 中国科学院过程工程研究所 一种不含表面活性剂的水包油乳液及其用途
WO2016016401A1 (en) * 2014-08-01 2016-02-04 Boehringer Ingelheim Vetmedica Gmbh Nanoparticles, methods of preparation, and uses thereof
CN108685873A (zh) * 2018-07-16 2018-10-23 中国医学科学院生物医学工程研究所 仿生型自组装球形核酸纳米颗粒及其制备方法与用途
CN108743939A (zh) * 2018-08-07 2018-11-06 中国医学科学院生物医学工程研究所 共载抗原、mpla与imq的阳离子磷脂-聚合物杂化纳米粒疫苗佐剂及制备方法与应用
CN108992666A (zh) * 2018-08-07 2018-12-14 中国医学科学院生物医学工程研究所 靶向共载抗原和tlr激动剂的阳离子磷脂-聚合物杂化纳米粒疫苗佐剂及制备方法与应用
CN110613844A (zh) * 2019-10-23 2019-12-27 中国医学科学院生物医学工程研究所 一种迷你联合佐剂纳米颗粒及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6608422B2 (ja) * 2014-03-25 2019-11-20 ザ ガバメント オブ ザ ユナイテッド ステイツ，アズ リプリゼンティッド バイ ザ セクレタリー オブ ジ アーミー モノホスホリルリピッドａ（ｍｐｌａ）含有リポソーム組成物およびサポニンを含む非毒性アジュバント製剤

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015192603A1 (zh) * 2014-06-18 2015-12-23 中国科学院过程工程研究所 一种不含表面活性剂的水包油乳液及其用途
WO2016016401A1 (en) * 2014-08-01 2016-02-04 Boehringer Ingelheim Vetmedica Gmbh Nanoparticles, methods of preparation, and uses thereof
CN108685873A (zh) * 2018-07-16 2018-10-23 中国医学科学院生物医学工程研究所 仿生型自组装球形核酸纳米颗粒及其制备方法与用途
CN108743939A (zh) * 2018-08-07 2018-11-06 中国医学科学院生物医学工程研究所 共载抗原、mpla与imq的阳离子磷脂-聚合物杂化纳米粒疫苗佐剂及制备方法与应用
CN108992666A (zh) * 2018-08-07 2018-12-14 中国医学科学院生物医学工程研究所 靶向共载抗原和tlr激动剂的阳离子磷脂-聚合物杂化纳米粒疫苗佐剂及制备方法与应用
CN110613844A (zh) * 2019-10-23 2019-12-27 中国医学科学院生物医学工程研究所 一种迷你联合佐剂纳米颗粒及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EUN-JU KO ET AL.: "MPL and CpG combination adjuvants promote homologous and heterosubtypic cross protection of inactivated split influenza virus vaccine", ANTIVIRAL RES ., vol. 156, 31 August 2018 (2018-08-31), pages 107 - 115, XP085416170, DOI: 10.1016/j.antiviral.2018.06.004 *
YOURI LEE ET AL.: "A unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus", VIROLOGY, vol. 534, 31 August 2019 (2019-08-31), pages 1 - 13, XP085733280, DOI: 10.1016/j.virol.2019.05.010 *

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