CN110585426B - 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗 - Google Patents

一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗 Download PDF

Info

Publication number
CN110585426B
CN110585426B CN201910766153.XA CN201910766153A CN110585426B CN 110585426 B CN110585426 B CN 110585426B CN 201910766153 A CN201910766153 A CN 201910766153A CN 110585426 B CN110585426 B CN 110585426B
Authority
CN
China
Prior art keywords
antigen
adjuvant
liver cancer
nano vaccine
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910766153.XA
Other languages
English (en)
Other versions
CN110585426A (zh
Inventor
张闯年
王晓莉
孔德领
孙洪范
裴萌月
徐蓉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Biomedical Engineering of CAMS and PUMC
Original Assignee
Institute of Biomedical Engineering of CAMS and PUMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Biomedical Engineering of CAMS and PUMC filed Critical Institute of Biomedical Engineering of CAMS and PUMC
Priority to CN201910766153.XA priority Critical patent/CN110585426B/zh
Publication of CN110585426A publication Critical patent/CN110585426A/zh
Application granted granted Critical
Publication of CN110585426B publication Critical patent/CN110585426B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001174—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • 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
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/844—Liver
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明涉及一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127‑136肽（GPC3127‑136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，载体材料、免疫抗原和佐剂之间的质量比是1:1～10:0.3～0.8。本发明的纳米疫苗能够增加树突状细胞（dendritic cell，DC）对抗原和佐剂的胞吞量，上调DC表面共刺激分子及促进TNF‑a、IL‑6等细胞因子分泌，利于激发机体产生有效的免疫应答，且其原料廉价易得、制备方法简单易重复、易于大规模加工生产，能够增强肝癌的免疫治疗效果，具有良好的应用前景。

Description

一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗
技术领域
本发明涉及生物医领域，尤其是涉及一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法。
背景技术
肝癌的发病率及死亡率在逐年增加，严重危害人类健康和安全。肝癌免疫治疗是通过重新激活机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。
肿瘤疫苗在肿瘤免疫治疗中占有重要地位，肿瘤疫苗经体内抗原提呈细胞处理、加工，并将抗原信息提呈给T细胞，从而激发机体免疫反应。树突状细胞（dendritic cell，DC）是体内已知功能最强的专职抗原提呈细胞，是启动、调控和维持免疫应答的中心环节。
寻找有效的抗原对于肿瘤疫苗的研发至关重要。磷脂酰肌醇蛋白聚糖3（Glypican3，GPC3）是一种肝癌胚胎蛋白，其在大多数原发性肝癌细胞中高表达，但在正常肝脏和良性肝脏病变组织中检测不到，因此，GPC3可以作为肿瘤抗原或治疗靶点。GPC-3127-136肽同样能够诱导产生细胞毒T细胞（CTL）反应，亦可作为肝癌特异性抗原，且多肽更易于合成，便于生产，纯度更高，价格也更低廉。
疫苗中的抗原以游离状态进入机体，会被体液稀释、酶降解，使得DC对抗原的摄取量极低，难以诱导机体产生有效的免疫应答。纳米疫苗可以保护抗原不被快速降解和清除，通过对纳米粒子结构进行设计还可以实现对抗原的靶向输送和控制释放，进而提高DC对抗原的摄取量、延长抗原的作用时间，利于激发机体产生有效的免疫应答。
聚乙烯亚胺（PEI）是一种水溶性聚合物,分子链上存在的伯、仲、叔胺基，使其呈碱性和阳离子活性,对阴离子物质有较好的亲和性和吸附性。天然多糖海藻酸钠（ALG）是一种线性阴离子多糖，具有良好的生物相容性和生物可降解性，对人体无毒，廉价易得，已在食品、医疗、生物技术和药学等领域得到了广泛的应用。疫苗佐剂(Adjuvant)是能够非特异性地改变或增强机体对抗原的特异性免疫应答。佐剂还能够诱发机体产生长期、高效的免疫反应，同时又能减少抗原的用量，降低肿瘤疫苗的生产成本。
现有的肿瘤疫苗对肿瘤的免疫治疗效果较低，且肿瘤疫苗的原料成本高，制备方法复杂，不利于大规模加工生产。
发明内容
本发明设计了一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法，其解决的技术问题是现有的肿瘤疫苗对肿瘤的免疫治疗效果较低，且肿瘤疫苗的原料成本高，制备方法复杂，不利于大规模加工生产。
为了解决上述存在的技术问题，本发明采用了以下方案：
一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽（GPC3127-136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8。
进一步，所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm。
进一步，所述海藻酸钠和所述聚乙烯亚胺之间的质量比是1:0.4～3。
进一步，所述海藻酸钠的分子量为8000-50000。
进一步，所述聚乙烯亚胺的分子量为3000-25000。
一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法，包括以下步骤：
步骤一，将GPC3127-136水溶液和聚乙烯亚胺水溶液混合得到PC3127-136和聚乙烯亚胺混合溶液，再将海藻酸钠水溶液和CpG水溶液混合得到海藻酸钠和CpG混合溶液；
步骤二，在搅拌条件下，将所述海藻酸钠和CpG混合溶液滴加到所述GPC3127-136和聚乙烯亚胺混合溶液中，继续搅拌得到所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗。
静电相互作用在步骤二中，海藻酸钠和CPG带负电，PEI和多肽带正电，滴加行程纳米粒原理就是静电相互作用。
进一步，步骤一中，所述GPC3127-136水溶液的浓度为0.5 mg/ml～4 mg/ml；所述聚乙烯亚胺水溶液的浓度为0.5 mg/ml～3 mg/ml；所述CpG水溶液的浓度为0.5 mg/ml～4mg/ml；所述海藻酸钠水溶液的浓度为0.5 mg/ml～2 mg/ml。
进一步，步骤一中，所述GPC3127-136水溶液和所述聚乙烯亚胺水溶液之间的质量比为1:0.05～0.5;所述海藻酸钠水溶液和所述CpG水溶液之间的质量比为1:0.8～1.5。
进一步，步骤二中，将所述海藻酸钠和CpG混合溶液滴加到所述GPC3127-136和聚乙烯亚胺混合溶液中后继续搅拌的时间为1min～15min。
该应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法具有以下有益效果：
（1）本发明的纳米疫苗能够增加树突状细胞（dendritic cell，DC）对抗原和佐剂的胞吞量，上调DC表面共刺激分子及促进TNF-a、IL-6等细胞因子分泌，利于激发机体产生有效的免疫应答，且其原料廉价易得、制备方法简单易重复、易于大规模加工生产，能够增强肝癌的免疫治疗效果，具有良好的应用前景。
（2）本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法中，纳米疫苗能够同时递送抗原和佐剂，同时能够增加DC对抗原和佐剂的胞吞量，从而提高肿瘤的免疫治疗效果。
（3）本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗及其制备方法中，制备的纳米粒子原料廉价易得、制备方法简单易重复、易于大规模加工生产，具有良好的应用前景。
附图说明
图1：本发明实施例1中得到的抗原和佐剂共递送纳米疫苗的粒径分布图；
图2：本发明实施例1中DC表面共刺激分子CD40、CD80以及CD86的荧光强度柱状图；
图3：本发明实施例2中得到的抗原和佐剂共递送纳米疫苗的粒径分布图；
图4：本发明实施例2中DC分泌的TNF-a、IL-6浓度柱状图；
图5：本发明实施例3中得到的荧光标记纳米疫苗的粒径分布图；
图6：本发明实施例3中DC对荧光标记纳米疫苗的胞吞共聚焦结果图。
具体实施方式
下面结合图1至图6，对本发明做进一步说明：
本发明应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽（GPC3127-136，AMFKNNYPSL）为免疫抗原，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8，海藻酸钠和聚乙烯亚胺之间的质量比是1:0.4～3，海藻酸钠的分子量为8000-50000，聚乙烯亚胺的分子量为3000-25000。
应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法，包括以下步骤：
步骤一，将GPC3127-136水溶液和聚乙烯亚胺水溶液混合得到PC3127-136和聚乙烯亚胺混合溶液，再将海藻酸钠水溶液和CpG水溶液混合得到海藻酸钠和CpG混合溶液，其中GPC3127-136水溶液的浓度为0.5 mg/ml～4 mg/ml；聚乙烯亚胺水溶液的浓度为0.5 mg/ml～3 mg/ml；CpG水溶液的浓度为0.5 mg/ml～4 mg/ml；海藻酸钠水溶液的浓度为0.5 mg/ml～2 mg/ml；GPC3127-136水溶液和聚乙烯亚胺水溶液之间的质量比为1:0.05～0.5;海藻酸钠水溶液和CpG水溶液之间的质量比为1:0.8～1.5。
步骤二，在搅拌条件下，将海藻酸钠和CpG混合溶液滴加到GPC3127-136和聚乙烯亚胺混合溶液中，继续搅拌1min～15min得到应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗。
实施例一：
纳米疫苗的制备：
将GPC3127-136水溶液（1mg/ml，0.5ml）和PEI水溶液（0.5mg/ml，0.2ml）混合，再将ALG水溶液（0.5mg/ml，0.5ml）和CpG水溶液（0.5mg/ml，0.5ml）混合，搅拌条件下，将ALG和CpG混合溶液滴加到GPC3127-136和PEI的混合溶液中，搅拌5min，得到抗原和佐剂共递送纳米疫苗。
如图1所示,本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为111.9nm。
纳米疫苗促DC成熟评价：
收集第六天未成熟的的DC，按106个细胞每孔，将DC铺于24孔板，贴壁2小时后，加入纳米疫苗，GPC3127-136浓度为10微克每毫升，继续培养48小时。轻吹细胞收集于离心管中，离心，细胞进行流式抗体标记，利用流式细胞仪检测DC表面共刺激分子CD40、CD80以及CD86的表达。
如图2所示，DC与纳米疫苗共培养后，DC表面共刺激分子CD40、CD80以及CD86产生的荧光强度更强，说明纳米疫苗对DC促成熟效果更好。
实施例二：
纳米疫苗的制备：
将GPC3127-136水溶液（2mg/ml，1ml）和PEI水溶液（2mg/ml，0.3ml）混合，再将ALG水溶液（2mg/ml，0.1ml）和CpG水溶液（2mg/ml，0.1ml）混合，搅拌条件下，将ALG和CpG混合溶液滴加到GPC3127-136和PEI的混合溶液中，搅拌2min，得到抗原和佐剂共递送纳米疫苗。
如图3所示，本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为173.7nm。
纳米疫苗促DC细胞因子分泌评价：
收集第六天未成熟的的DC，按106个细胞每孔，将DC铺于24孔板，贴壁2小时后，加入纳米疫苗，GPC3127-136浓度为10微克每毫升，继续培养48小时。轻吹细胞收集于离心管中，离心，收集上清液，酶联免疫吸附测定（ELISA）检测上清液中TNF-a、IL-6的含量，图4是DC分泌的TNF-a、IL-6浓度柱状图。
如图4所示，DC与纳米疫苗共培养后，DC分泌的TNF-a、IL-6含量较与游离抗原和佐剂组明显增多，说明纳米疫苗刺激DC细胞因子分泌效果更好。
实施例三：
荧光标记纳米疫苗的制备：
将异硫氰酸荧光素标记的GPC3127-136水溶液（4mg/ml，2ml）和PEI水溶液（0.5mg/ml，1ml）混合，再将ALG水溶液（2mg/ml，0.2ml）和罗丹明荧光素标记的CpG水溶液（1mg/ml，0.5ml）混合，搅拌条件下，将ALG和罗丹明荧光素标记的CpG混合溶液滴加到异硫氰酸荧光素标记的GPC3127-136和PEI的混合溶液中，搅拌3min，得到抗原和佐剂共递送荧光标记纳米疫苗。
如图5所示，本实施例获得的纳米疫苗的粒径分布比较均一，平均粒径为224nm。
DC对荧光标记纳米疫苗的胞吞评价：
按106个细胞每皿，将DC铺于共聚焦皿中，贴壁24小时后，加入含有荧光标记的纳米疫苗的培养基，GPC3127-136浓度为10微克每毫升，再培养24小时。用PBS洗3次细胞，多聚甲醛固定，Lamp-1染溶酶体，DAPI染细胞核，用共聚焦显微镜观察DC对荧光标记纳米疫苗的胞吞共聚焦结果。
如图6所示，纳米疫苗能够同时增加DC对多肽抗原和佐剂的胞吞量，有利于提高免疫响应。
上面结合附图对本发明进行了示例性的描述，显然本发明的实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (1)

1.一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗，其特征在于：以海藻酸钠和聚乙烯亚胺为载体材料，以肝癌特异性多肽抗原磷脂酰肌醇蛋白聚糖3127-136肽GPC3127-136为免疫抗原，所述肽GPC3127-136的序列为AMFKNNYPSL，以CpG寡脱氧核苷酸为佐剂，通过静电相互作用制备的抗原和佐剂共递送纳米疫苗，其中，所述载体材料、所述免疫抗原和所述佐剂之间的质量比是1:1～10:0.3～0.8；
所述应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的粒径为100nm～1000nm；
所述海藻酸钠和所述聚乙烯亚胺之间的质量比是1:0.4～3；
所述海藻酸钠的分子量为8000-50000；
所述聚乙烯亚胺的分子量为3000-25000。
CN201910766153.XA 2019-08-19 2019-08-19 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗 Active CN110585426B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910766153.XA CN110585426B (zh) 2019-08-19 2019-08-19 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910766153.XA CN110585426B (zh) 2019-08-19 2019-08-19 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗

Publications (2)

Publication Number Publication Date
CN110585426A CN110585426A (zh) 2019-12-20
CN110585426B true CN110585426B (zh) 2023-03-21

Family

ID=68854698

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910766153.XA Active CN110585426B (zh) 2019-08-19 2019-08-19 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗

Country Status (1)

Country Link
CN (1) CN110585426B (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110585425B (zh) * 2019-08-19 2023-03-21 中国医学科学院生物医学工程研究所 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法
CN117122677A (zh) * 2020-04-24 2023-11-28 苏州维益生物科技有限公司 含有至少2个硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗
CN114366808B (zh) * 2021-12-14 2023-10-13 南京农业大学 一种多糖和病毒抗原共递送纳米疫苗、其制备方法及应用
CN114807229B (zh) * 2022-05-27 2024-09-24 中国科学院长春应用化学研究所 细胞膜、纳米疫苗及其制备方法和应用
CN115998864A (zh) * 2022-11-23 2023-04-25 华中科技大学协和深圳医院 一种负载免疫佐剂的黑磷光热纳米疫苗及其制备方法和在癌症治疗中的应用
CN116650629A (zh) * 2023-06-14 2023-08-29 首都医科大学附属北京佑安医院 一种小鼠树突状细胞外泌体介导的肝癌疫苗的制备

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108785666A (zh) * 2018-06-05 2018-11-13 中国医学科学院生物医学工程研究所 一种应用于肝癌免疫治疗的dc靶向性纳米疫苗及其制备方法
CN109998998A (zh) * 2019-04-19 2019-07-12 中国科学院长春应用化学研究所 一种纳米疫苗及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11191821B2 (en) * 2016-02-27 2021-12-07 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Peptide vaccine formulations and use thereof for inducing an immune response

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108785666A (zh) * 2018-06-05 2018-11-13 中国医学科学院生物医学工程研究所 一种应用于肝癌免疫治疗的dc靶向性纳米疫苗及其制备方法
CN109998998A (zh) * 2019-04-19 2019-07-12 中国科学院长春应用化学研究所 一种纳米疫苗及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Identification of an H2-Kb or H2-Db restricted and glypican-3-derived cytotoxic T-lymphocyte epitope peptide;Iwama T et al.;《Int J Oncol》;20130123;第42卷(第3期);摘要、第837页左栏末段 *
Infection-mimicking materials to program dendritic cells in situ;O.A. Ali, et al.;《Nat. Mater.》;20090111;第8卷(第2期);全文 *

Also Published As

Publication number Publication date
CN110585426A (zh) 2019-12-20

Similar Documents

Publication Publication Date Title
CN110585426B (zh) 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗
Sokolova et al. The use of calcium phosphate nanoparticles encapsulating Toll-like receptor ligands and the antigen hemagglutinin to induce dendritic cell maturation and T cell activation
Varypataki et al. Cationic liposomes loaded with a synthetic long peptide and poly (I: C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity
Pradhan et al. The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma
De Geest et al. Surface‐engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function
Kasturi et al. Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles
Sokolova et al. Quantitative determination of the composition of multi-shell calcium phosphate–oligonucleotide nanoparticles and their application for the activation of dendritic cells
Beaudette et al. In vivo studies on the effect of co-encapsulation of CpG DNA and antigen in acid-degradable microparticle vaccines
Fischer et al. The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles
CN108434122B (zh) 聚电解质杂化中空二氧化硅纳米颗粒及其制备方法与应用
CN107456575A (zh) 一种二氧化锰纳米佐剂及其制备方法、应用
CN112516297B (zh) 一种基于鱼精蛋白为载体的抗原和佐剂共传递纳米疫苗的制备方法及其应用
Jiang et al. Lymph node delivery strategy enables the activation of cytotoxic T lymphocytes and natural killer cells to augment cancer immunotherapy
Cheng et al. Polyethylenimine-mediated CpG oligodeoxynucleotide delivery stimulates bifurcated cytokine induction
CN110613844A (zh) 一种迷你联合佐剂纳米颗粒及其制备方法和应用
CN113274492B (zh) 一种基于羟基氧化铝纳米羧基改性的复合疫苗佐剂的制备方法
CN113528436B (zh) 基于淋巴细胞的同源靶向性人工抗原呈递细胞及其构建和应用
CN112089834B (zh) 基于氧化石墨烯的茯苓多糖纳米佐剂及佐剂/抗原共递送疫苗的制备与应用
CN110585425B (zh) 一种应用于肝癌免疫治疗的抗原和佐剂共递送纳米疫苗的制备方法
Jiao et al. Lentinan-functionalized graphene oxide hydrogel as a sustained antigen delivery system for vaccines
Zhang et al. Pickering emulsion stabilized by Chinese Yam polysaccharides PLGA for enhanced humoral and cellular immune responses
CN114712493B (zh) 一种疫苗递送载体及其制备方法和应用
CN118591632A (zh) 细菌外膜囊泡来源的核酸纳米疫苗及其应用
Salvador et al. Dendritic cells interactions with the immune system–Implications for vaccine development
CN110623938B (zh) 一种mpc修饰的树状大分子包裹纳米金颗粒及其制备和应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant