CN113456832B - 一种转铁蛋白修饰的包载抗体的纳米粒及其应用 - Google Patents

一种转铁蛋白修饰的包载抗体的纳米粒及其应用 Download PDF

Info

Publication number
CN113456832B
CN113456832B CN202110718389.3A CN202110718389A CN113456832B CN 113456832 B CN113456832 B CN 113456832B CN 202110718389 A CN202110718389 A CN 202110718389A CN 113456832 B CN113456832 B CN 113456832B
Authority
CN
China
Prior art keywords
transferrin
bcr
antibody
abl
nanoparticles
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
CN202110718389.3A
Other languages
English (en)
Other versions
CN113456832A (zh
Inventor
冯文莉
姜国云
袁颖
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.)
International Institute Of In Vitro Diagnostics Chongqing Medical University
Original Assignee
International Institute Of In Vitro Diagnostics Chongqing Medical University
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 International Institute Of In Vitro Diagnostics Chongqing Medical University filed Critical International Institute Of In Vitro Diagnostics Chongqing Medical University
Priority to CN202110718389.3A priority Critical patent/CN113456832B/zh
Publication of CN113456832A publication Critical patent/CN113456832A/zh
Application granted granted Critical
Publication of CN113456832B publication Critical patent/CN113456832B/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
    • 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
    • A61K47/6935—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 otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/62—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 non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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

Abstract

本发明公开了一种转铁蛋白修饰的包载抗体的纳米粒，是将靶向BCR/ABL蛋白的特异性抗体包载到PLGA纳米颗粒内，然后添加转铁蛋白修饰纳米颗粒表面得到的。还公开了其制备方法和其在制备用于慢性髓系白血病靶向治疗的药物中的应用。本申请采用转铁蛋白(Tf)修饰的聚乳酸‑羟基乙酸共聚物(PLGA)纳米颗粒将特异性识别BCR/ABL致病蛋白的抗体递送到慢粒细胞中，靶向结合BCR/ABL致病蛋白，并通过泛素‑蛋白酶体降解途径引起BCR/ABL蛋白的快速降解，起到对慢粒细胞的靶向杀伤效果，经实验证实，制备的纳米颗粒对慢粒细胞的靶向杀伤效果显著。

Description

一种转铁蛋白修饰的包载抗体的纳米粒及其应用
技术领域
本发明涉及靶向给药技术领域，具体涉及一种转铁蛋白修饰的包载抗体的纳米粒及其应用。
背景技术
慢性髓细胞白血病，简称慢粒，是一种源于骨髓造血干细胞的恶性增殖性疾病，其发病率为1-2/100000，约占成人白血病新诊断病例的15-20％。慢性髓细胞白血病存在t(9；22)(q32；q21)易位的Ph染色体，从而形成bcr/abl癌基因并编码具有高酪氨酸激酶活性的BCR/ABL癌蛋白，BCR/ABL癌蛋白能够激活细胞内一系列下游信号通路，凋亡受阻。酪氨酸激酶抑制剂类药物(TKI)出现使慢粒的治疗进入了新的时期，现已成为慢粒病人的首选治疗药物。但是，由于BCR/ABL突变导致的病人对TKI的耐药其临床应用同样受到挑战。目前存在有15-25％的患者对TKI耐药或者不耐受，因此，新的慢粒治疗方法的出现对TKI耐药的病人有重要意义。
发明内容
本申请发明人采用转铁蛋白(Tf)修饰的聚乳酸-羟基乙酸共聚物(PLGA)纳米颗粒将特异性识别BCR/ABL致病蛋白的抗体递送到慢粒细胞中(图1为本发明的纳米颗粒实验抗体细胞内递送过程示意图)，靶向结合BCR/ABL致病蛋白，并通过泛素-蛋白酶体降解途径引起BCR/ABL蛋白的快速降解，起到对慢粒细胞的靶向杀伤效果，经实验证实，制备的纳米颗粒对慢粒细胞的靶向杀伤效果显著。基于此，本发明要求保护如下技术方案：
一种转铁蛋白修饰的包载抗体的纳米粒，是将靶向BCR/ABL蛋白的特异性抗体包载到PLGA纳米颗粒内，然后添加转铁蛋白修饰纳米颗粒表面得到的。
在上述技术方案中，是采用乳化溶剂挥发法将靶向BCR/ABL蛋白的特异性抗体包载到PLGA纳米颗粒上的。
所述靶向BCR/ABL蛋白的特异性抗体为抗BCR/ABL抗体。
上述的转铁蛋白修饰的包载抗体的纳米粒的制备方法，包括如下步骤：
1)采用乳化溶剂挥发法将靶向BCR/ABL蛋白的特异性抗体包载到PLGA纳米颗粒上；
2)将步骤1)得到的纳米颗粒与转铁蛋白溶液混匀后振荡过夜，离心、洗涤、收集米颗粒，即得。
所述步骤1)具体过程是：称量PLGA并溶解于三氯甲烷中作为油相，加入香豆素6溶液作为荧光染料，将anti-BCR/ABL抗体和NH4HCO3溶解于超纯水中作为水相，将水相加入油相中混匀，超声得到油包水颗粒；加入聚乙烯醇溶液后超声、磁力搅拌挥发有机溶剂后，离心30min、洗涤并收集纳米颗粒。
所述PLGA与anti-BCR/ABL抗体的质量比为18～22﹕0.5，优选20﹕0.5；
所述PLGA与步骤2)所用的转铁蛋白的质量比为18～22﹕10，优选20﹕10。
在上述技术方案中，将水相加入油相中混匀后超声2min，40％Amp，每10s间歇5s；
加入聚乙烯醇溶液后超声6min，40％Amp，每20s间歇10s；
所述离心为4℃低温超速离心机13300rpm离心30min。
本发明还保护上述的转铁蛋白修饰的包载抗体的纳米粒在制备用于慢性髓系白血病靶向治疗的药物中的应用。
本发明还保护一种药物，包含上述的转铁蛋白修饰的包载抗体的纳米粒以及药学上可接受的载体。
本发明的有益效果是：
1.慢粒的治疗手段主要为使用TKI降低慢粒BCR/ABL的酪氨酸激酶活性，但是由于病人会出现TKI耐药及不耐受等问题，使得TKI应用受限。本发明将抗BCR/ABL抗体与泛素-蛋白酶体系统结合起来，可直接降解慢粒细胞内BCR/ABL癌蛋白。因此，本发明对TKI耐药的慢粒细胞同样有效，也对慢粒病人的治疗提供了一种有临床应用潜能的治疗方法。
2.本发明采用PLGA纳米颗粒作为抗BCR/ABL抗体细胞内递送的载体，打破了传统治疗性抗体不能穿透细胞膜入胞结合致病蛋白的局限性，可将抗BCR/ABL抗体有效的递送至CML细胞内部。其中PLGA纳米颗粒在体内可分解为二氧化碳和水，具有安全无毒高效等优点。
3.本发明采用转铁蛋白对PLGA纳米颗粒表面进行修饰，增强了纳米颗粒的慢粒细胞靶向效果。
附图说明
图1为本发明的纳米颗粒实验抗体细胞内递送过程示意图。
图2为Ab@Tf-Cou6-PLGANPs合成流程示意图。
图3为Ab@Tf-Cou6-PLGANPs的表征图，其中，(A)透射电镜照片；(B)纳米颗粒粒径分布图；(C)纳米颗粒电位图；(D)纳米颗粒释放率；(E)纳米颗粒成分验证；(F)溶血实验检测纳米颗粒生物相容性；(G)纳米颗粒的蛋白吸附能力。
图4为Ab@Tf-Cou6-PLGANPs的细胞摄取情况，其中，(A)纳米颗粒在不同时间的细胞摄取流式结果；(B)纳米颗粒在不同剂量下的细胞摄取流式结果；(C)流式细胞术检测转铁蛋白修饰对纳米细胞摄取的影响；(D)纳米颗粒的细胞摄取机制。
图5为Ab@Tf-Cou6-PLGANPs对慢粒细胞株K562和K562/G01增殖及凋亡的影响，其中，(A)纳米颗粒对慢粒细胞增殖能力的影响；(B)纳米颗粒对慢粒细胞克隆形成能力的影响；(C)流式细胞术检测纳米颗粒对慢粒细胞凋亡的影响；(D)免疫印迹实验检测凋亡分子表达情况；(E)纳米颗粒对非慢粒细胞增殖能力的影响。
图6为纳米颗粒对病人标本凋亡的影响，其中，(A)慢粒病人；(B)非慢粒供者。
图7为纳米颗粒对慢粒模型小鼠的治疗效果，其中，(A)外周血白细胞计数；(B)小鼠肝脏重量；(C)小鼠脾脏重量；(D)小鼠体重起始变化；(E)小鼠骨髓CD45+细胞比例；(F)小鼠骨髓瑞氏染色结果；(G)小鼠生存期曲线；(H)纳米颗粒对小鼠的肝肾毒性。
具体实施方式
下面结合实施例对本发明作进一步说明，但并不因此而限制本发明。
下述实施例中的实验方法，如无特别说明，均为常规方法。
主要试剂及其来源：
anti-BCR/ABL抗体：品牌：上海生工，货号：D120191
转铁蛋白：品牌：索莱宝Solarbio，货号:T8010
香豆素6：CAS号38215-36-0
人慢性粒细胞白血病细胞株：K562细胞
慢粒细胞的伊马替尼耐药细胞系：K562/G01细胞
人肺腺癌细胞：A549细胞，重庆医科大学检验学院实验室保存
白血病细胞株：HL-60、NB4细胞：重庆医科大学检验医学院实验室保存
实施例1
一、Ab@Tf-Cou6-PLGANPs的制备
利用乳化溶剂挥发法合成(流程示意图如图2所示)：精准称量20.0mgPLGA(聚乳酸-羟基乙酸共聚物)并溶解于2ml三氯甲烷中作为油相，加入10μl香豆素6溶液(0.05％)作为荧光染料，将0.5mg anti-BCR/ABL抗体和0.25mgNH4HCO3溶解于200μl超纯水中作为水相。将水相缓缓加入油相中混匀，超声2min(40％Amp，每10s间歇5s)得到油包水颗粒。加入10mlPVA(聚乙烯醇)溶液(1％)并超声6min(40％Amp，每20s间歇10s)，磁力搅拌4h(750rpm)挥发有机溶剂后，4℃低温超速离心机13300rpm离心30min，洗涤并收集纳米颗粒(记为Ab@Cou6-PLGANPs)。将得到的纳米颗粒与2ml转铁蛋白(transferrin)溶液(5mg/ml)混匀后4℃振荡过夜，4℃低温超速离心机13300rpm离心30min洗涤并收集纳米颗粒，得到经转铁蛋白修饰的包载anti-BCR/ABL抗体的PLGA纳米颗粒，记为Ab@Tf-Cou6-PLGANPs。制备未加anti-BCR/ABL抗体、但加了转铁蛋白的纳米颗粒作为对照，记为Tf-Cou6-PLGANPs。
二、纳米颗粒的表征
TEM和DLS检测：将合成的纳米颗粒Ab@Tf-Cou6-PLGANPs稀释到合适浓度后送TEM和DLS检测对其进行粒径和电位的表征。TEM结果显示纳米颗粒Ab@Tf-Cou6-PLGANPs呈球形(如图3A)；DLS结果显示Ab@Tf-Cou6-PLGANPs大小均一，粒径在290nm左右，电位在-13mV左右(图3B、3C)。
纳米颗粒释放率检测：将获得的纳米颗粒Ab@Tf-Cou6-PLGANPs分别溶解于1ml pH＝7.4和pH＝5.0的PBS中，37℃振荡(100rpm)，分别于24h，48h,72h,96h,120h,144h,168h时取出上清后补充1ml PBS。将取得的上清用增强BCA法测得每24h蛋白释放量并计算累计释放率。图3D结果显示纳米颗粒在pH＝7.4的中性环境下7天释放率为38％左右，而在pH＝5.0的酸性环境中7天释放率达到65％左右，说明纳米颗粒在酸性条件下释放率更高。
纳米颗粒成分验证：将转铁蛋白，anti-BCR/ABL抗体，Ab@Tf-Cou6-PLGANPs进行SDS-PAGE电泳，考马斯亮蓝染色后，利用脱色液脱色，Bio-Rad凝胶成像仪拍照并比较条带。图3E结果显示Ab@Tf-Cou6-PLGANPs组在转铁蛋白及抗体相应位置有同样条带，说明转铁蛋白及抗体成功包载于纳米颗粒上。
纳米颗粒生物相容性检测：取新鲜Nod-Scid小鼠血液2ml，2000rpm离心10min，用生理盐水洗涤红细胞至上清无色，将红细胞以2％的压积重悬，将0.8ml的红细胞悬液与0.2ml超纯水(阳性对照)、0.2ml生理盐水(阴性对照)及0.2ml不同浓度的Ab@Tf-Cou6-PLGANPs、PLGANPs、Tf-Cou6-PLGANPs纳米颗粒混匀后37℃放置2h。2000rpm离心15min后加入96孔板中，酶标仪540nm处检测吸光度，计算溶血率。溶血实验结果显示4种浓度(0.125、0.25、0.5、1.0mg/ml)的纳米颗粒溶血率都很低，说明纳米颗粒具有良好的生物相容性(图3F)。
蛋白吸附实验：将纳米颗粒PLGANPs、Tf-Cou6-PLGANPs、Ab@Tf-Cou6-PLGANPs分别以0.5mg/ml的浓度溶解于pH＝7.4及pH＝5.0的PBS溶液中，将纳米颗粒溶液与牛血清白蛋白(0.25mg/ml)在37℃水浴振荡2h后，13300rpm离心30min后取上清，利用BCA法检测蛋白浓度并计算纳米颗粒的蛋白吸附情况。蛋白吸附实验结果显示几种纳米颗粒的蛋白吸附能力均较低，预示纳米颗粒具有良好的稳定性(图3G)。
三、纳米颗粒的细胞摄取
纳米颗粒的细胞摄取：将Ab@Tf-Cou6-PLGANPs及Ab@Cou6-PLGANPs分别同慢粒细胞共孵育一定时间后，预冷PBS洗涤，0.4ml PBS重悬后使用BD C5流式检测细胞荧光强度；将相同浓度的Ab@Tf-Cou6-PLGANPs与慢粒细胞共培养不同时间后离心并用PBS洗涤，将收到的细胞用0.4mlPBS重悬后使用BD C5流式细胞仪检测；将不同浓度的Ab@Tf-Cou6-PLGANPs与慢粒细胞共培养一定时间后离心并用PBS洗涤，将收到的细胞用0.4mlPBS重悬后使用BD C5流式细胞仪检测。结果显示转铁蛋白修饰可以提高细胞对纳米颗粒的摄取(图4D)且纳米颗粒的细胞摄取呈时间及剂量依赖性(图4A,4B)。图4中，K562为伊马替尼敏感株，K562/G01为伊马替尼耐药株。
纳米颗粒细胞摄取机制验证：将慢粒细胞分为三组处理①无蔗糖，37℃②450mM蔗糖，37℃③无蔗糖，4℃，三组处理1h后，加入纳米颗粒Ab@Tf-Cou6-PLGANPs继续处理1h，将细胞离心洗涤后加入0.4ml PBS重悬上BD C5流式细胞仪检测。结果显示加入胞吞抑制剂蔗糖后细胞的纳米颗粒摄取明显减弱，表明纳米颗粒的细胞摄取通过胞吞作用进行(图4C)。
四、纳米颗粒对慢粒细胞株的作用：
检测PLGANPs、Tf-Cou6-PLGANPs、Ab@Tf-Cou6-PLGANPs纳米颗粒对慢粒细胞株的作用，同时设置阴性对照，检测结果如下：
(1)细胞增殖能力检测：将慢粒细胞铺至96孔板中，分别加入纳米颗粒处理，在0h，24h、48h、72h时分别加入10ul CCK-8溶液，37℃放置3h后酶联免疫检测仪450nm处测量吸光度。结果显示Ab@Tf-Cou6-PLGANPs处理组的细胞增殖能力明显减弱，说明Ab@Tf-Cou6-PLGANPs具有抑制CML细胞增殖的作用(图5A)。
(2)细胞克隆形成能力检测:将慢粒细胞铺至96孔板中，加入纳米颗粒处理并在37℃孵箱放置1-2周后，倒置显微镜下观察细胞克隆形成情况。实验结果显示经Ab@Tf-Cou6-PLGA NPs处理组细胞的克隆形成数量及大小均小于对照组，表明Ab@Tf-Cou6-PLGANPs可抑制CML细胞的克隆形成能力(图5B)。
(3)细胞凋亡检测：将纳米颗粒与慢粒细胞共处理一定时间后，收集细胞，用流式测凋亡试剂盒染色后上流式细胞仪检测细胞凋亡；将纳米颗粒与慢粒细胞共处理一定时间后，收集细胞并提取总蛋白，westernblot检测PARP及Caspase-3的蛋白水平变化。流式结果显示Ab@Tf-Cou6-PLGANPs处理组CML细胞的凋亡率明显增加(图5C)；实验结果显示Ab@Tf-Cou6-PLGA NPs处理组PARP及Caspase-3的蛋白裂带明显，表明Ab@Tf-Cou6-PLGANPs有促进CML细胞凋亡的作用(图5D)。
(4)纳米颗粒对非慢粒细胞增殖能力的影响：将A549、HL-60、NB4细胞铺至96孔板中，加入纳米颗粒处理，在0h，24h、48h、72h时分别加入10ulCCK-8溶液，37℃放置3h后酶联免疫检测仪450nm处测量吸光度。结果显示纳米颗粒对细胞增殖能力无明显影响，说明Ab@Tf-Cou6-PLGANPs对非慢粒细胞增殖能力无影响(图5E)。
五、纳米颗粒对慢粒病人标本的作用：
慢粒病人标本凋亡检测：将纳米颗粒(PLGANPs、Ab@Tf-Cou6-PLGANPs，设置阴性对照)与慢粒细胞共处理一定时间后，收集细胞用流式测凋亡试剂盒染色后上流式细胞仪检测细胞凋亡。结果显示Ab@Tf-Cou6-PLGANPs处理可促进慢粒病人细胞凋亡(图6A)，而对正常人细胞无明显影响(图6B)。
实施例2、纳米颗粒对慢粒小鼠模型的作用
一、慢粒小鼠模型构建
利用X射线对小鼠进行亚致死量辐射，剂量为250cGY。收集K562/G01细胞对小鼠进行尾静脉注射，每只5×106个K562/G01细胞，注射体积为200ul。在造瘤完成1周后将小鼠按5只一组随机分组，分别注射PBS及纳米颗粒(PLGANPs、Tf-Cou6-PLGANPs、Ab@Tf-Cou6-PLGANPs，同时设置阴性对照)，注射体积为200ul，每周注射一次。
二、小鼠成瘤能力检测
造瘤完成3周后，定期观察小鼠生存状态，定期称量小鼠体重，检测小鼠外周血白细胞数量。当小鼠体重极度下降，出现毛色暗沉精神萎靡，弓背跛行等症状时处死小鼠。取出小鼠肝脾进行拍照称重；取出小鼠骨髓染色进行骨髓细胞学检查。
结果显示Ab@Tf-Cou6-PLGANPs处理组小鼠外周血白细胞计数明显低于其他组(图7A)；Ab@Tf-Cou6-PLGANPs处理组小鼠肝脾重量也显著低于其他组小鼠(图7B、7C)；小鼠实验前后体重对比结果显示Ab@Tf-Cou6-PLGANPs处理组小鼠体重只有轻微下降，而对照组小鼠体重下降较为严重(7D)；小鼠骨髓流式结果显示Ab@Tf-Cou6-PLGANPs处理组小鼠骨髓的人CD45+细胞比例明显低于其他组(7E)；图7F显示Ab@Tf-Cou6-PLGA NPs处理组小鼠骨髓中慢粒细胞浸润明显低于其他组；图7G小鼠生存曲线显示Ab@Tf-Cou6-PLGA NPs处理组小鼠具有更长的生存期。以上结果均表明Ab@Tf-Cou6-PLGANPs处理在小鼠体内发挥了良好的抗肿瘤生成能力。
三、纳米颗粒毒性作用检测
将纳米颗粒通过尾静脉注射入小鼠体内，检测小鼠外周血ALT、AST、BUN水平。图7H显示纳米颗粒具有良好的生物安全性，几种纳米颗粒对小鼠的肝肾功能均无损伤。

Claims (8)

1.一种转铁蛋白修饰的包载抗体的纳米粒，其特征在于：是将靶向BCR/ABL蛋白的特异性抗体抗BCR/ABL抗体包载到PLGA纳米颗粒内，然后添加转铁蛋白修饰纳米颗粒表面得到的。
2.如权利要求1所述的转铁蛋白修饰的包载抗体的纳米粒，其特征在于：是采用乳化溶剂挥发法将靶向BCR/ABL蛋白的特异性抗体包载到PLGA纳米颗粒上的。
3.如权利要求1或2所述的转铁蛋白修饰的包载抗体的纳米粒的制备方法，其特征在于，包括如下步骤：
1）采用乳化溶剂挥发法将靶向BCR/ABL蛋白的特异性抗体包载到PLGA
纳米颗粒上；
2）将步骤1）得到的纳米颗粒与转铁蛋白溶液混匀后振荡过夜，离心、洗
涤、收集纳米颗粒，即得。
4.如权利要求3所述的方法，其特征在于：所述步骤1）具体过程是：称量 PLGA并溶解于三氯甲烷中作为油相，加入香豆素6溶液作为荧光染料，将 anti-BCR/ABL抗体和 NH4HCO3 溶解于超纯水中作为水相，将水相加入油相中混匀，超声得到油包水颗粒；加入聚乙烯醇溶液后超声、磁力搅拌挥发有机溶剂后，离心30min、洗涤并收集纳米颗粒。
5.如权利要求4所述的方法，其特征在于：所述PLGA与anti-BCR/ABL抗体的质量比为18～22﹕0.5；
所述PLGA与步骤2）所用的转铁蛋白的质量比为18～22﹕10。
6.如权利要求4所述的方法，其特征在于：
将水相加入油相中混匀后超声2min，40%Amp，每10s间歇5s；
加入聚乙烯醇溶液后超声6min，40%Amp，每20s间歇10s；
所述离心为4℃低温超速离心机13300rpm离心30min。
7.权利要求1或2所述的转铁蛋白修饰的包载抗体的纳米粒在制备用于慢性髓系白血病靶向治疗的药物中的应用。
8.一种药物，其特征在于：包含权利要求1或2所述的转铁蛋白修饰的包载抗体的纳米粒以及药学上可接受的载体。
CN202110718389.3A 2021-06-28 2021-06-28 一种转铁蛋白修饰的包载抗体的纳米粒及其应用 Active CN113456832B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110718389.3A CN113456832B (zh) 2021-06-28 2021-06-28 一种转铁蛋白修饰的包载抗体的纳米粒及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110718389.3A CN113456832B (zh) 2021-06-28 2021-06-28 一种转铁蛋白修饰的包载抗体的纳米粒及其应用

Publications (2)

Publication Number Publication Date
CN113456832A CN113456832A (zh) 2021-10-01
CN113456832B true CN113456832B (zh) 2023-06-30

Family

ID=77873324

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110718389.3A Active CN113456832B (zh) 2021-06-28 2021-06-28 一种转铁蛋白修饰的包载抗体的纳米粒及其应用

Country Status (1)

Country Link
CN (1) CN113456832B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116531524B (zh) * 2023-07-07 2023-09-15 深圳大学总医院 一种脑靶向纳米粒及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003200882A1 (en) * 1995-01-05 2003-06-12 The Board Of Regents Acting For And On Behalf Of The University Of Michigan Surface-modified nanoparticles and method of making and using same
CN101123983A (zh) * 2004-10-27 2008-02-13 米迪缪尼股份有限公司 特定修饰对相关抗原的亲和力来调节抗体的特异性
WO2008026224A2 (en) * 2006-08-31 2008-03-06 University Of Hyderabad Novel nanoparticles of apotransferrin / transferrin, pharmaceutical composition containing them and processes for their preparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003200882A1 (en) * 1995-01-05 2003-06-12 The Board Of Regents Acting For And On Behalf Of The University Of Michigan Surface-modified nanoparticles and method of making and using same
CN101123983A (zh) * 2004-10-27 2008-02-13 米迪缪尼股份有限公司 特定修饰对相关抗原的亲和力来调节抗体的特异性
WO2008026224A2 (en) * 2006-08-31 2008-03-06 University Of Hyderabad Novel nanoparticles of apotransferrin / transferrin, pharmaceutical composition containing them and processes for their preparation

Also Published As

Publication number Publication date
CN113456832A (zh) 2021-10-01

Similar Documents

Publication Publication Date Title
US9351931B2 (en) Pharmaceutical preparation for tumor chemotherapy and method for producing the same
CN108543074A (zh) 一种用于肿瘤治疗的外泌体包裹的纳米载药系统及其制备
CN108377643A (zh) 使用超小纳米粒子通过铁死亡诱导营养素剥夺癌细胞的细胞死亡的治疗方法
Li et al. Nanomedicine-based cancer immunotherapies developed by reprogramming tumor-associated macrophages
EP3525802B1 (en) Cancer stem cell exosomes
CN108403658A (zh) 细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用
Zhao et al. Biomimetic nanotherapy: core–shell structured nanocomplexes based on the neutrophil membrane for targeted therapy of lymphoma
Chen et al. A DNA/DMXAA/Metal–Organic Framework Activator of Innate Immunity for Boosting Anticancer Immunity
Li et al. PD-L1-targeted microbubbles loaded with docetaxel produce a synergistic effect for the treatment of lung cancer under ultrasound irradiation
Deng et al. Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells
CN113456832B (zh) 一种转铁蛋白修饰的包载抗体的纳米粒及其应用
CN113018438A (zh) Cxcr2抑制剂在制备治疗鼻咽癌的药物中的用途
Dai et al. Tumor-targeted biomimetic nanoplatform precisely integrates photodynamic therapy and autophagy inhibition for collaborative treatment of oral cancer
CN114404571A (zh) 一种装载化疗药物且tigit过表达的工程化载药细胞膜囊泡以及制备方法和应用
Zhu et al. Low-dose X-ray radiodynamic therapy solely based on gold nanoclusters for efficient treatment of deep hypoxic solid tumors combined with enhanced antitumor immune response
Wu et al. Time rules the efficacy of immune checkpoint inhibitors in photodynamic therapy
CN111732635B (zh) 与cd123蛋白特异性结合的多肽、多肽复合物、共递送系统及其制备方法与应用
Wang et al. Multifunctional nano-realgar hydrogel for enhanced glioblastoma synergistic chemotherapy and radiotherapy: a new paradigm of an old drug
Li et al. Engineering Lipusu with lysophosphatidylcholine for improved tumor cellular uptake and anticancer efficacy
CN106103701A (zh) 改造的自然杀手细胞及其组成与用途
Wang et al. Multi-ligand modified PC@ DOX-PA/EGCG micelles effectively inhibit the growth of ER+, PR+ or HER 2+ breast cancer
Dreicer et al. A phase II trial of deferoxamine in patients with hormone-refractory metastatic prostate cancer
Xu et al. Sendai virus acts as a nano-booster to excite dendritic cells for enhancing the efficacy of CD47-directed immune checkpoint inhibitors against breast carcinoma
CN113521035B (zh) 化学免疫联合治疗纳米药物的制备方法及应用
CN113398275B (zh) 一种用于光动力治疗的细菌载体及其制备方法与应用

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