CN103212083B - Method for preparing stable albumin nano-particles - Google Patents

Method for preparing stable albumin nano-particles Download PDF

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CN103212083B
CN103212083B CN 201310124591 CN201310124591A CN103212083B CN 103212083 B CN103212083 B CN 103212083B CN 201310124591 CN201310124591 CN 201310124591 CN 201310124591 A CN201310124591 A CN 201310124591A CN 103212083 B CN103212083 B CN 103212083B
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albumin
nanoparticles
pharmacologically active
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method
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CN103212083A (en
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王文坦
黄延宾
程易
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清华大学
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Abstract

一种制备稳定的白蛋白纳米颗粒的方法,属于生物医药材料制备技术领域。 A method of stabilizing albumin nanoparticles prepared, the field of biotechnology pharmaceutical preparation materials. 该方法利用无生物毒性的谷胱甘肽或半胱氨酸对白蛋白进行预处理,打开分子内二硫键,采用醇类等反溶剂沉淀白蛋白,利用巯基-二硫键交换反应得到分子间二硫键的白蛋白纳米颗粒。 The method utilizes non-cysteine ​​glutathione or toxicity of pre-albumin, open an intramolecular disulfide bond, using an anti-solvent such as alcohols albumin precipitation by sulfhydryl - disulfide exchange reaction between the molecules to give disulfide albumin nanoparticles. 利用所制备的纳米粒可以用于体内递送药理活性物质和/或诊断助剂。 Nanoparticles can be prepared for use in vivo delivery of pharmacologically active substances and / or diagnostic aid. 本发明所得到的白蛋白纳米颗粒的优点在于不但在稀释条件下具有良好的稳定性,同时也在还原型环境中具有氧化还原响应。 The advantage of the present invention is obtained in that not only the albumin nanoparticles have good stability under dilute conditions, but also in an environment having a reduced oxidation-reduction response. 这种特性使得该颗粒可以稳定存在于生物体内血液循环系统中,并在细胞中在还原型谷胱甘肽的作用下降解并释放出所包裹的药物。 This property makes the particles may be stable in the blood circulatory system in vivo, and the degradation effects of reduced glutathione in the cell and release of the entrapped drug.

Description

一种制备稳定的白蛋白纳米颗粒的方法 A method of stabilizing albumin nanoparticles prepared

技术领域 FIELD

[0001] 本发明涉及一种白蛋白纳米颗粒的制备方法,属于生物医药材料制备技术领域。 [0001] The present invention relates to a process for the preparation of albumin nanoparticles, the field of biotechnology pharmaceutical preparation materials.

背景技术: Background technique:

[0002] 白蛋白是一种生物内源性蛋白,具有的可生物降解、无毒、无抗原性等诸多特点, 被认为是一个理想的药物载体,白蛋白载药体系是现今药学研究中一个极具生命力的研究方向。 [0002] Albumin is the one biogenic protein with biodegradable, non-toxic, non-antigenic and many other features, is considered to be an ideal drug carrier, drug delivery systems is now albumin contained in a pharmaceutical research great vitality of the research. 白蛋白载药体系主要分为两大类,一类是化学偶联的白蛋白载药体系,另一类是物理结合的白蛋白载药体系。 Albumin drug system is mainly divided into two categories, one is chemically conjugated albumin drug system, another system albumin drug physically bonded. 化学偶联的白蛋白可以改善药物的药代动力学特性,但结合后形成了新的化学分子。 Chemical coupling of albumin can improve pharmacokinetic properties of the drug, but to form a new chemical molecules. 物理结合形式的白蛋白给药体系是一个更为理想的载药模式,将药物分子包裹在白蛋白纳米颗粒中,可以明显提高非水溶性药物在水溶液中(即体内血液循环系统中)的稳定性与溶解度。 Physical delivery systems albumin-bound form is a more preferable mode of the drug, the drug molecules encapsulated in albumin nanoparticles can significantly improve the stability of the water-insoluble drugs in an aqueous solution (i.e., the blood circulation system) of sex and solubility. 同时,利用肿瘤组织的透过性增强及滞留效应(EPR effect), 可以使得白蛋白纳米载药体系达到靶向给药的目的。 Meanwhile, enhanced permeability and retention effect (EPR effect) using tumor tissue, may cause the nano-albumin drug targeting system for administration purposes.

[0003] 由于白蛋白分子具有极好的水溶性,如何使得白蛋白纳米颗粒在水中有着良好的稳定性,在稀释条件下不溶解是目前制备技术上的难点。 [0003] Since the albumin molecule having excellent water solubility, how to make albumin nanoparticles have good stability in water, does not dissolve under dilute conditions is prepared technical difficulties. 戊二醛等交联剂常被用来稳定得到的纳米颗粒,但是戊二醛会非选择性的结合白蛋白表面的氨基位点,在生物体内会释放出醛类残基,对生物体有着显著毒副作用。 Crosslinking agent such as glutaraldehyde are often used to stabilize the nanoparticles obtained, but will be non-selective glutaraldehyde amino albumin binding site surface, the in vivo release aldehyde residues with the organism significant side effects. 有报道一种自组装的白蛋白纳米粒合成技术(CN102048695A),利用巯基乙醇等作为还原剂打开了白蛋白分子的二硫键,使其利用疏水相互作用结合成为纳米颗粒,但该颗粒在10%乙醇水溶液中即全部溶解,说明该颗粒仅靠疏水相互作用稳定,而并非二硫键结合稳定(Gong MG et al.,Biomacromolecules, 2012, 1 3, 23-28) aAmerican Bioscience公司开发了一种基于二硫键形成的nab技术(Nanoparticle albumin-bound technology),以白蛋白作为基质和稳定剂,在不加入任何乳化剂和交联剂的情况下制得到白蛋白纳米粒(US6753006B1)。 It reported a self-assembled nanoparticle albumin synthesis (CN102048695A), using mercaptoethanol as a reducing agent to open the disulfide albumin molecule, so that the use of nanoparticles become bound hydrophobic interactions, but the particles 10 % aqueous solution of ethanol, i.e. completely dissolved, indicating that the particles are stabilized only by hydrophobic interaction, rather than a disulfide bond stabilized (Gong MG et al., Biomacromolecules, 2012, 1 3, 23-28) aAmerican Bioscience has developed a based nab technology (nanoparticle albumin-bound technology) disulfide bond formation, as albumin stabilizer and a matrix prepared to give albumin nanoparticles (US6753006B1) without the addition of any emulsifying agent and a crosslinking agent. 但近年来在对Abraxane产品稀释实验(Chauhan VP et al.,Nature Nanotech·,2012, 7, 383-388.表明,Abraxane 稀释到缓冲溶液或者牛血清白蛋白溶液中后,会立刻分解成为1 〇nm左右的大小的颗粒,这表明该产品也并非由二硫键交联而得。 In recent years Abraxane product after dilution experiments (Chauhan VP et al., Nature Nanotech ·, 2012, 7, 383-388. Show, Abraxane diluted into a buffer solution or a solution of bovine serum albumin, will immediately break down into a square particle size of about nm, which indicates that the product is not obtained by crosslinking by disulfide bonds.

发明内容: SUMMARY:

[0004] 本发明的目的是提供一种制备稳定的白蛋白纳米颗粒的方法,该白蛋白纳米粒由分子间二硫键交联而稳定,用以解决某些药理活性物质难以进行体内递送的问题。 [0004] The object of the present invention is to provide a process for preparing stabilized nanoparticle albumin method, while the albumin nanoparticles stabilized by intermolecular disulfide crosslinked to solve some of the pharmacologically active substance is difficult to perform in vivo delivery problem.

[0005] 为了实现本发明的目的,本发明的技术方案为: [0005] To achieve the object of the present invention, the technical solution of the present invention is:

[0006] -种制备稳定的白蛋白纳米颗粒的方法,如下步骤进行: [0006] - A method of preparing a stable species of albumin nanoparticles, the following steps:

[0007] 1)制备体积质量浓度为10〜200mg/mL的白蛋白水溶液; [0007] 1) Preparation of a volume concentration of the aqueous solution of albumin 10~200mg / mL of;

[0008] 2)在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; [0008] 2) added to the aqueous solution of albumin in glutathione or cysteine ​​so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min;

[0009] 3)在步骤2)反应后的溶液中加入乙醇或叔丁醇,在温度10〜60°C下,反应5〜 120min,得到分子间二硫键交联的白蛋白纳米颗粒的悬浊液;所述的白蛋白水溶液与加入的乙醇或叔丁醇的体积比为1 :1〜5 ; [0009] 3) Add ethanol or t-butanol in the solution after the step 2) reaction at a temperature of 10~60 ° C, the reaction 5~ 120min, albumin nanoparticle suspension obtained intermolecular disulfide crosslinked turbid liquid; the volume of the aqueous solution of albumin was added ethanol or t-butanol ratio of 1: 1 ~ 5;

[0010] 4)将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到白蛋白纳米颗粒溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到白蛋白纳米颗粒。 [0010] 4) The suspension of albumin nanoparticles at a temperature of 0~20 ° C dialysis, to give albumin nanoparticle solution, and then freeze-dried, spray dried or dehydrated by distillation under reduced pressure, to give albumin nano particles.

[0011] 一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,按如下步骤进行: [0011] A preparation method of delivering a pharmacologically active agent in vivo for albumin nanoparticles, proceed as follows:

[0012] 1)制备体积质量浓度为10〜200mg/mL的白蛋白水溶液; [0012] 1) Preparation of a volume concentration of the aqueous solution of albumin 10~200mg / mL of;

[0013] 2)在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; [0013] 2) added to the aqueous solution of albumin in glutathione or cysteine ​​so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min;

[0014] 3)在步骤2)反应后的溶液中加入药理活性药物的乙醇溶液或药理活性药物的叔丁醇溶液,在温度10〜60°c下反应5〜120min,得到包裹药理活性物质的白蛋白纳米颗粒的悬浊液;所述的白蛋白水溶液与加入的药理活性药物的乙醇溶液或药理活性药物的叔丁醇溶液的体积比为1 :1〜5 ; [0014] 3) in step 2) t was added to the reaction solution after the pharmacologically active agent in ethanol or a butanol solution pharmacologically active agent, at a temperature of 10~60 ° c under reaction 5~120min, wrapping the pharmacologically active substance to give suspension of nanoparticles of albumin; aqueous ethanol solution of the albumin with a pharmacologically active drug or pharmacologically active agent is added in a volume ratio of tert-butanol solution is 1: 1 ~ 5;

[0015] 4)将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到包裹药理活性物质的白蛋白纳米颗粒的溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到包裹药理活性物质的白蛋白纳米颗粒。 [0015] 4) The suspension of nanoparticles of albumin in the dialysis at a temperature of 0~20 ° C to give a solution of albumin nanoparticles package pharmacologically active substance, then freeze-dried, spray-drying or distillation under reduced pressure dehydration to give a nanoparticle albumin wrapped pharmacologically active substance.

[0016] 本发明技术方案中得到的白蛋白纳米粒平均粒径为10〜500nm。 The average particle diameter of albumin nanoparticles Technical Solution [0016] The present invention is 10~500nm.

[0017] 本发明技术方案中所述的白蛋白分子包括人血清白蛋白(human serum albumin, HSA),牛血清白蛋白(bovine serum albumin,BSA),重组人血清白蛋白(recombinant HSA, rHSA),或它们的组合。 [0017] The albumin molecule aspect of the present invention include human serum albumin (human serum albumin, HSA), bovine serum albumin (bovine serum albumin, BSA), recombinant human serum albumin (recombinant HSA, rHSA) , or a combination thereof.

[0018] 本发明技术方案中所述的包裹药理活性物质的白蛋白纳米颗粒中可包载占纳米粒总量1 %〜90%的药理活性物质。 [0018] aspect of the present invention in the albumin nanoparticle wrapped pharmacologically active substance can be entrapped in total account for 1% ~ 90% of the nanoparticles pharmacologically active substance.

[0019] 本发明技术方案中所述的药理活性物质为抗癌药物。 [0019] aspect of the present invention in the pharmacologically active substance is an anticancer drug.

[0020] 本发明技术方案中所述的抗癌药物包括紫杉醇、多西紫杉醇、阿霉素、姜黄素、钼、 甲氨蝶呤或它们的组合。 [0020] In the aspect of the present invention, anticancer drugs include paclitaxel, docetaxel, doxorubicin, curcumin, molybdenum, methotrexate, or a combination thereof.

[0021] 本发明所制得的白蛋白纳米颗粒和包裹药理活性物质的白蛋白纳米颗粒的优点在于:纳米颗粒中的白蛋白有分子间二硫键交联,在pH范围3-4以及6-10之间的水溶液中均稳定存在,在体积分数为1〜50%的乙醇水溶液中稳定存在,并在生理条件(37°C,pH = 7.4)下的稀释实验中稳定存在,同时该颗粒可以在还原性环境中能够逐步溶解。 [0021] The advantages of the present invention is prepared nanoparticle albumin and albumin nanoparticles package pharmacologically active substance that: albumin nanoparticles have an intermolecular disulfide bond crosslinked, at a pH range of 3-4 and 6 in aqueous solution between -10 are stable, stable in volume fraction of 1~50% aqueous ethanol, and diluted in the experiment under stable in physiological conditions (7.4 37 ° C, pH =), while the granules It may be capable of gradually dissolving in a reducing environment. 以上特性使得该颗粒可以稳定存在于生物体内血液循环系统中,并在细胞中在还原型谷胱甘肽的作用下降解并释放出所包裹的药物。 More characteristics such that the particles can be stably present in the blood circulatory system in vivo, and the degradation effects of reduced glutathione in the cell and release of the entrapped drug.

附图说明: BRIEF DESCRIPTION OF:

[0022] 图1为本发明中白蛋白纳米粒在不同溶剂环境中的稳定性研究。 [0022] FIG 1 Stability of nanoparticles of albumin in different solvents environment of the present invention.

[0023] 图2为本发明中白蛋白纳米粒在还原型条件下逐步溶解后的SDS-PAGE电泳图。 [0023] Figure 2 SDS-PAGE of albumin nanoparticles view of the gradual reduction in the dissolved condition of the present invention. 其中,1为白蛋白纳米粒的条带,2为白蛋白低聚物的条带,3为白蛋白二聚体和三聚体的条带,4为白蛋白单分子的条带。 Wherein a strip of albumin nanoparticles, 2 albumin oligomer strip, a strip 3 of albumin dimers and trimers tape, strip 4 is a single molecule of albumin.

[0024] 图3为本发明中白蛋白-姜黄素纳米粒的扫面电子显微镜图像和透射电子显微镜图像。 Curcumin scan electron microscope image of the surface of the nanoparticles and TEM images - [0024] FIG albumin present invention. 其中,A和B为扫面电子显微镜图像,C和D为透射电子显微镜图像。 Wherein, A and B are scanning electron microscope images, C and D are transmission electron microscope images.

具体实施方式: Detailed ways:

[0025] 本发明提供的一种制备稳定的白蛋白纳米颗粒的方法,该方法按如下步骤进行: [0025] a method of stabilizing albumin nanoparticles prepared according to the present invention provided, the method proceeds as follows:

[0026] 1)制备体积质量浓度为10〜200mg/mL的白蛋白水溶液;所述的白蛋白一般采用人血清白蛋白、牛血清白蛋白、重组人血清白蛋白或它们的组合。 [0026] 1) Preparation of aqueous albumin solution by volume concentration of 10~200mg / mL; said albumin is generally used as human serum albumin, bovine serum albumin, recombinant human serum albumin, or a combination thereof.

[0027] 2)在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; [0027] 2) added to the aqueous solution of albumin in glutathione or cysteine ​​so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min;

[0028] 3)在步骤2)反应后的溶液中加入乙醇或叔丁醇,在温度10〜60°C下反应5〜 120min,得到分子间二硫键交联的白蛋白纳米颗粒的悬浊液;所述的白蛋白水溶液与加入的乙醇或叔丁醇的体积比为1 :1〜5 ; [0028] 3) Add ethanol or t-butanol in the solution of step 2) the reaction, the reaction 5~ 120min at a temperature of 10~60 ° C, to obtain a suspension of albumin nanoparticles intermolecular disulfide crosslinked solution; volume of said aqueous albumin solution with the addition of ethanol or tert-butanol ratio of 1: 1 ~ 5;

[0029] 4)将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到白蛋白纳米颗粒溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到白蛋白纳米颗粒。 [0029] 4) The suspension of albumin nanoparticles at a temperature of 0~20 ° C dialysis, to give albumin nanoparticle solution, and then freeze-dried, spray dried or dehydrated by distillation under reduced pressure, to give albumin nano particles. 得到的白蛋白纳米粒平均粒径为10〜500nm。 Albumin nanoparticles obtained average particle diameter of 10~500nm.

[0030] 本发明提供的一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,该方法按如下步骤进行: [0030] The present invention provides a method of preparing nanoparticle albumin for delivering a pharmacologically active agent in vivo, the method proceeds as follows:

[0031] 1)制备体积质量浓度为10〜200mg/mL的白蛋白水溶液; [0031] 1) Preparation of a volume concentration of the aqueous solution of albumin 10~200mg / mL of;

[0032] 2)在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; [0032] 2) added to the aqueous solution of albumin in glutathione or cysteine ​​so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min;

[0033] 3)在步骤2)反应后的溶液中加入药理活性药物的乙醇溶液或活性药物的叔丁醇溶液,在温度10〜60°C下反应5〜120min,得到包裹药理活性物质的白蛋白纳米颗粒的悬浊液;所述的白蛋白水溶液与加入的药理活性药物的乙醇溶液或药理活性药物的叔丁醇溶液的体积比为1 :1〜5 ;药理活性物质为抗癌药物,例如紫杉醇、多西紫杉醇、阿霉素、姜黄素、钼及钼的类似物、甲氨蝶呤或它们的组合。 [0033] 3) in step 2) t was added to the reaction solution after the pharmacologically active agent in ethanol or a butanol solution of the active agent, at a temperature of 10~60 ° C reaction 5~120min, to give a white wrapping pharmacologically active substance protein nanoparticles of the suspension; t albumin and an aqueous solution of the pharmacologically active agent is added to a solution in ethanol or butanol pharmacologically active agent solution volume ratio of 1: 1 ~ 5; pharmacologically active substance is an anticancer drug, such as paclitaxel, docetaxel, doxorubicin, curcumin, molybdenum and molybdenum analogues, methotrexate, or a combination thereof.

[0034] 4)将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到包裹药理活性物质的白蛋白纳米颗粒的溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到包裹药理活性物质的白蛋白纳米颗粒。 [0034] 4) The suspension of nanoparticles of albumin in the dialysis at a temperature of 0~20 ° C to give a solution of albumin nanoparticles package pharmacologically active substance, then freeze-dried, spray-drying or distillation under reduced pressure dehydration to give a nanoparticle albumin wrapped pharmacologically active substance. 白蛋白纳米粒中包载的药理活性物质占纳米粒总总量的1%〜90%。 Albumin entrapped in nanoparticles of pharmacologically active substance in a proportion of 1% ~ 90% of the total amount of nanoparticles.

[0035] 为了更好的理解本发明,下面结合五个实例对本发明做进一步详细说明。 [0035] For a better understanding of the invention, in conjunction with the following five examples of the present invention will be further described in detail. 但是本发明保护范围不局限于实施例所表达的范围。 However, the scope of the present invention is not limited to the scope of the expressed embodiment.

[0036] 下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。 [0036] In the following examples the experimental procedure, if no special instructions, all conventional methods; the reagents and materials, as no special instructions, are available from commercial sources.

[0037] 实施例1,制备二硫键稳定的人血清白蛋白纳米颗粒。 [0037] Example 1. Preparation of disulfide-stabilized human serum albumin nanoparticles.

[0038] 以人血清白蛋白与还原型谷胱:甘肤为原料,制备-硫键稳定的人血清白蛋白纳米颗粒。 [0038] In human serum albumin and reduced glutathione: Gan skin prepared starting - sulfur bonds stable human serum albumin nanoparticles.

[0039] 具体制备方法如下: [0039] DETAILED prepared as follows:

[0040] 将人血清白蛋白20mg溶于lmL的水溶液中,并加入20mM还原型谷胱甘肽,室温下反应1小时,然后加入4mL无水乙醇反应30分钟,随后将所制得的悬浊液至于透析袋中,在4°C的环境下的去离子水中透析24小时,即得到二硫键稳定的人血清白蛋白纳米颗粒,而且白蛋白纳米粒的平均粒径为100〜300nm(ZetaPALS, Zeta Potential Analyzer)。 [0040] The human serum albumin solution 20mg dissolved in lmL, 20mM reduced glutathione was added and reacted for 1 hour at room temperature, followed by addition of 4mL of anhydrous ethanol for 30 minutes, then the suspension obtained as the dialysis bag was dialyzed for 24 hours in deionized water at 4 ° C environment, i.e., to give disulfide-stabilized human serum albumin nanoparticles, nanoparticle albumin and an average particle diameter of 100~300nm (ZetaPALS , Zeta Potential Analyzer).

[0041] 所制得的纳米颗粒在不同溶剂环境中的稳定性可以从图1中看出。 [0041] The nanoparticles prepared in various solvents stable environment can be seen in FIG. 所制得的纳米颗粒在还原环境下的逐步溶解过程可以从图2的SDS-PAGE凝胶电泳图中得到确认。 The resulting nanoparticles can be confirmed from FIG SDS-PAGE gel electrophoresis of FIG. 2 gradual dissolution process in a reducing environment.

[0042] 实施例2,制备_硫键稳定的人血清白蛋白纳米颗粒。 [0042] Example 2. Preparation of sulfur bonds _ stable human serum albumin nanoparticles.

[0043] 以人血清白蛋白与还原型谷胱甘肽为原料,制备二硫键稳定的人血清白蛋白纳米颗粒。 [0043] In human serum albumin and glutathione prepared starting disulfide-stabilized human serum albumin nanoparticles.

[0044] 具体制备方法如下: [0044] DETAILED prepared as follows:

[0045] 将人血清白蛋白200mg溶于lmL的水溶液中,并加入500mM还原型谷胱甘肽,50°C 反应10分钟,然后加入lmL无水乙醇反应5分钟,然后将所制得的悬浊液至于透析袋中,在l〇°C的环境下的去离子水中透析24小时,即得到二硫键稳定的人血清白蛋白纳米颗粒,而且白蛋白纳米粒的平均粒径为100〜400nm。 [0045] The human serum albumin solution dissolved in lmL of 200mg and added 500mM glutathione, 50 ° C for 10 minutes, followed by addition of lmL of absolute ethanol for 5 minutes, then the suspension obtained as a dialysis bag turbid solution, dialyzed in deionized water for 24 hours at ambient l〇 ° C., to obtain the disulfide-stabilized human serum albumin nanoparticles, nanoparticle albumin and an average particle diameter of 100~400nm .

[0046] 实施例3,制备二硫键稳定的姜黄素-人血清白蛋白纳米颗粒载药体系。 Human serum albumin nanoparticle drug carrier systems - [0046] 3. Preparation of disulfide-stabilized curcumin embodiment.

[0047] 以人血清白蛋白,还原型谷胱甘肽和姜黄素为原料,制备二硫键稳定的人血清白蛋白包覆姜黄素的纳米颗粒载药体系。 [0047] In human serum albumin, glutathione and curcumin product was prepared disulfide-stabilized human serum albumin-coated nanoparticles curcumin drug system.

[0048] 具体制备方法如下: [0048] DETAILED prepared as follows:

[0049] 将人血清白蛋白40mg溶于lmL的水溶液中,并加入50mM还原型谷胱甘肽,室温下反应1小时,然后加入4mL的3mg/mL姜黄素乙醇溶液反应30分钟,随后将所制得的悬浊液至于透析袋中,在4°C的环境下的去离子水中透析24小时,即得到二硫键稳定的姜黄素-人血清白蛋白包纳米颗粒。 [0049] The human serum albumin solution 40mg dissolved in lmL, 50mM reduced glutathione was added and reacted for 1 hour at room temperature, and then 3mg / mL curcumin 4mL of ethanol was added for 30 minutes, followed by the As suspension prepared dialysis bag, dialyzed in deionized water for 24 hours at 4 ° C at ambient, i.e., to obtain a stable disulfide curcumin - human serum albumin-coated nanoparticles. 而且纳米粒的平均粒径为50〜400nm(ZetaPALS, Zeta Potential Analyzer),用反相C18住测定姜黄素的载药量,流动相为甲醇:水:乙酸(80 :19 :1),检测波长为428nm。 And an average particle size of nanoparticles 50~400nm (ZetaPALS, Zeta Potential Analyzer), live curcumin drug loading was measured by reverse-phase C18, mobile phase was methanol: water: acetic acid (80: 19: 1), detection wavelength to 428nm. HPLC分析表明本实验的姜黄素载药量达到21 %。 HPLC analysis indicated the drug loading of curcumin 21% in this experiment. 所制得的纳米颗粒形貌从图3的电镜图像得到确认。 The morphology of the nanoparticles prepared was confirmed from the SEM image of FIG. 3.

[0050] 实施例4,制备二硫键稳定的姜黄素-人血清白蛋白纳米颗粒载药体系。 Human serum albumin nanoparticle drug carrier systems - [0050] 4, prepared in Example disulfide stabilized curcumin.

[0051] 以人血清白蛋白,还原型谷胱甘肽和姜黄素为原料,制备二硫键稳定的人血清白蛋白包覆姜黄素的纳米颗粒载药体系。 [0051] In human serum albumin, glutathione and curcumin product was prepared disulfide-stabilized human serum albumin-coated nanoparticles curcumin drug system.

[0052] 具体制备方法如下: [0052] DETAILED prepared as follows:

[0053] 将人血清白蛋白10mg溶于lmL的水溶液中,并加入50mM还原型谷胱甘肽,室温下反应1小时,然后加入4mL的3mg/mL姜黄素乙醇溶液反应30分钟,随后将所制得的悬浊液至于透析袋中,在4°C的环境下的去离子水中透析24小时,即得到二硫键稳定的姜黄素-人血清白蛋白包纳米颗粒。 [0053] The human serum albumin solution 10mg dissolved in lmL, 50mM reduced glutathione was added and reacted for 1 hour at room temperature, and then 3mg / mL curcumin 4mL of ethanol was added for 30 minutes, followed by the As suspension prepared dialysis bag, dialyzed in deionized water for 24 hours at 4 ° C at ambient, i.e., to obtain a stable disulfide curcumin - human serum albumin-coated nanoparticles. 所制得的纳米粒的平均粒径为50〜400nm,HPLC分析表明本实验的姜黄素载药量达到40%。 The average particle diameter of the obtained nanoparticles is 50~400nm, HPLC analysis showed curcumin drug load of 40% in this experiment.

[0054] 实施例5,制备二硫键稳定的紫杉醇-人血清白蛋白纳米颗粒。 Human serum albumin nanoparticle - [0054] Example 5 Preparation of disulfide-stabilized paclitaxel embodiment.

[0055] 以人血清白蛋白,还原型谷胱甘肽和紫杉醇为原料,制备二硫键稳定的紫杉醇-人血清白蛋白纳米颗粒载药体系。 [0055] In human serum albumin, glutathione and paclitaxel is prepared starting disulfide-stabilized paclitaxel - human serum albumin nanoparticle drug system.

[0056] 具体制备方法如下: [0056] DETAILED prepared as follows:

[0057] 将人血清白蛋白40mg溶于lmL的水溶液中,并加入50mM还原型谷胱甘肽,25°C下反应4小时,然后加入3. 5mL的2. 5mg/mL紫杉醇的叔丁醇溶液反应30分钟,随后将所制得的悬浊液至于透析袋中,在4°C的环境下的去离子水中透析24小时,即得到二硫键稳定的紫杉醇-人血清白蛋白包纳米颗粒。 [0057] The human serum albumin solution 40mg dissolved in lmL, 50mM reduced glutathione was added and reacted for 4 hours at 25 ° C, followed by addition of 2. 5mg 3. 5mL / mL paclitaxel in tert-butanol solution was reacted for 30 minutes, then the suspension prepared as for dialysis bag and dialyzed for 24 hours in deionized water at 4 ° C environment, disulfide-stabilized paclitaxel that is obtained - human serum albumin-coated nanoparticles . 所制得的纳米粒的平均粒径为50〜400nm,HPLC分析表明本实验的紫杉醇载药量达到10%。 The average particle diameter of the obtained nanoparticles is 50~400nm, HPLC analysis revealed paclitaxel drug load of 10% in this experiment.

Claims (7)

1. 一种制备稳定的白蛋白纳米颗粒的方法,其特征在于该方法按如下步骤进行: 1) 制备体积质量浓度为10〜200mg/mL的白蛋白水溶液; 2) 在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; 3) 在步骤2)反应后的溶液中加入乙醇或叔丁醇,在温度10〜60°C下反应5〜120min, 得到分子间二硫键交联的白蛋白纳米颗粒的悬浊液;所述的白蛋白水溶液与加入的乙醇或叔丁醇的体积比为1 :1〜5 ; 4) 将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到白蛋白纳米颗粒溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到白蛋白纳米颗粒。 CLAIMS 1. A stabilized albumin nanoparticles prepared, wherein the method proceeds as follows: 1) Preparation of aqueous albumin solution by volume concentration of 10~200mg / mL; and 2) was added an aqueous solution of albumin in the valleys glutathione or cysteine, so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min; 3) in step 2) the reaction solution was added ethanol or tert-butanol, the reaction 5~120min at 10~60 ° C, to obtain a suspension of nanoparticles of albumin crosslinked intermolecular disulfide; said albumin aqueous solution was added ethanol or t-butanol the volume ratio of 1: 1 ~ 5; 4) the suspension of albumin nanoparticles at a temperature of 0~20 ° C dialysis, to give albumin nanoparticle solution, and then freeze-dried, spray-drying or distillation under reduced pressure dehydration treatment, to obtain a nanoparticle albumin.
2. 根据权利要求1所述的一种制备稳定的白蛋白纳米颗粒的方法,其特征在于:所述的白蛋白纳米粒平均粒径为10〜500nm。 2. A method for preparing a stabilized albumin nanoparticles according to claim 1, wherein: the average particle diameter of albumin nanoparticle 10~500nm.
3. 根据权利要求1所述的一种制备稳定的白蛋白纳米颗粒的方法,其特征在于:所述的白蛋白包括人血清白蛋白、牛血清白蛋白、重组人血清白蛋白或它们的组合。 The method of preparing a stabilized albumin nanoparticles according to claim 1, wherein: said albumin include human serum albumin, bovine serum albumin, recombinant human serum albumin or a combination thereof .
4. 一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,其特征在于该方法按如下步骤进行: 1) 制备体积质量浓度为10〜200mg/mL的白蛋白水溶液; 2) 在白蛋白水溶液中加入谷胱甘肽或半胱氨酸,使其在白蛋白水溶液中的摩尔浓度达到10〜500mM,在温度10〜60°C下反应5〜240min ; 3) 在步骤2)反应后的溶液中加入药理活性药物的乙醇溶液或活性药物的叔丁醇溶液,在温度10〜60°C下反应5〜120min,得到包裹药理活性物质的白蛋白纳米颗粒的悬池液;所述的白蛋白水溶液与加入的药理活性药物的乙醇溶液或药理活性药物的叔丁醇溶液的体积比为1 :1〜5 ; 4) 将白蛋白纳米颗粒的悬浊液在温度0〜20°C下透析,得到包裹药理活性物质的白蛋白纳米颗粒的溶液,再经过冷冻干燥、喷雾干燥或减压蒸馏进行脱水处理,得到包裹药理活性物质的白蛋白纳米颗粒。 4. A preparation method of delivering a pharmacologically active agent in vivo for albumin nanoparticle, wherein the method proceeds as follows: 1) Preparation of aqueous albumin solution by volume concentration of 10~200mg / mL; and 2) albumin was added an aqueous solution of cysteine ​​or glutathione, so that the molar concentration of albumin in the aqueous solution reaches 10~500mM, reacted at a temperature of 10~60 ° C 5~240min; 3) in step 2) the reaction after pharmacologically active agent was added in ethanol or t-butanol solution of the active agent, at a temperature of 10~60 ° C reaction 5~120min, cell suspension obtained was wrapped albumin nanoparticles of pharmacologically active substance; the albumin aqueous solution was added to an ethanol solution of the pharmacologically active drug or pharmacologically active agent is tert-butanol solution of a volume ratio of 1: 1 ~ 5; 4) the suspension of albumin nanoparticles at a temperature 0~20 ° C dialyzed to obtain a solution of albumin nanoparticles package pharmacologically active substance, then freeze-dried, spray dried or dehydrated by distillation under reduced pressure, to give the pharmacologically active substance wrapped albumin nanoparticles.
5. 根据权利要求4所述的一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,其特征在于:所述的白蛋白纳米粒中包载的药理活性物质占纳米粒总量的1%〜 90%。 5. According to one of claim 4 to the production method of delivering pharmacologically active agent in vivo for albumin nanoparticles, wherein: the nanoparticle albumin entrapped pharmacologically active substance accounted total of nanoparticles 1% to 90%.
6. 根据权利要求4所述的一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,其特征在于:所述的药理活性物质为抗癌药物。 6. A method according to claim 4, wherein the preparation method of delivering a pharmacologically active agent in vivo for albumin nanoparticles, wherein: the pharmacologically active substance is an anticancer drug.
7. 根据权利要求6所述的一种用于递送体内药理活性药物的白蛋白纳米颗粒的制备方法,其特征在于:所述的抗癌药物包括紫杉醇、多西紫杉醇、阿霉素、姜黄素、钼、甲氨蝶呤或它们的组合。 The one of the production method of claim 6 delivering pharmacologically active agent in vivo for albumin nanoparticles, wherein: the anticancer drugs include paclitaxel, docetaxel, doxorubicin, curcumin , molybdenum, methotrexate, or a combination thereof.
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