本申请是申请日为2010年08月09日，申请号为201010247885.7，发明创造名称为“一种制备用于体内递送药理活性物质的蛋白纳米粒的方法”的分案申请。This application is a divisional application with an application date of August 9, 2010, an application number of 201010247885.7, and an invention titled "A Method for Preparing Protein Nanoparticles for In vivo Delivery of Pharmacologically Active Substances".
技术领域technical field
本发明涉及一种制备用于体内递送药理活性物质的蛋白纳米粒的方法，属于药理活性物质的体内递送及其临床应用领域。具体来说，本发明是一种利用蛋白及多肽展开与再折叠或自组装的方法将药理活性物质包入蛋白，形成纳米粒的方法。The invention relates to a method for preparing protein nanoparticles for delivering pharmacologically active substances in vivo, and belongs to the field of in vivo delivery of pharmacologically active substances and their clinical applications. Specifically, the present invention is a method for encapsulating pharmacologically active substances into proteins by unfolding and refolding or self-assembling methods of proteins and polypeptides to form nanoparticles.
背景技术Background technique
静脉注射给药能够快速直接地作用于机体。为了降低静脉注射给药的副作用，有效的方法之一就是将药理活性物质包入微米或纳米级的粒子中。一方面，静脉注射的粒子可以缓释药理活性物质并延长药理活性物质的半衰期；另一方面，靶向材料可以将粒子中的药理活性物质靶向地释放。Intravenous administration can quickly and directly act on the body. In order to reduce the side effects of intravenous administration, one of the effective methods is to encapsulate pharmacologically active substances into micron or nanoscale particles. On the one hand, the intravenously injected particles can release the pharmacologically active substances slowly and prolong the half-life of the pharmacologically active substances; on the other hand, the targeting material can release the pharmacologically active substances in the particles in a targeted manner.
在现有技术涉及的蛋白粒子的制备方法中，如Abraxane®的制备方法（美国专利No.6,749,868，美国专利No.5,560,933），将蛋白溶液与有机溶剂均匀混合形成乳化液，高压匀浆，形成以紫杉醇为核心的白蛋白纳米粒。除了这种制备方法比较复杂之外，还需要用高压及相应的高温来去除有机溶剂，从而获得纳米粒。二氯甲烷，乙腈等有机溶剂具有毒性，需要控制其残留量。此外，这些方法包载药理活性物质的能力是有限的。同时，由于制备过程中的高压和剪切力，蛋白和多肽还可能失去生物活性。In the preparation method of protein particles involved in the prior art, such as the preparation method of Abraxane® (US Patent No. 6,749,868, US Patent No. 5,560,933), the protein solution is uniformly mixed with an organic solvent to form an emulsion, and homogenized under high pressure to form Albumin nanoparticles with paclitaxel as the core. In addition to the complexity of this preparation method, it is necessary to use high pressure and corresponding high temperature to remove the organic solvent, so as to obtain nanoparticles. Organic solvents such as dichloromethane and acetonitrile are toxic, and their residual amount needs to be controlled. Furthermore, these methods are limited in their ability to entrap pharmacologically active substances. At the same time, proteins and peptides may also lose their biological activity due to the high pressure and shear force during the preparation process.
蛋白粒子还被众多文献报道作为药理活性物质和诊断试剂的载体。其中，白蛋白微球可以用热交联和化学交联的方法来制备。热交联微球是在100～150℃下从乳化混合物（如白蛋白、需包载的药理活性物质和适当的油相）中制备。然后微球用适当的溶剂洗涤并保存。Leucuta等报道了热交联微球的制备方法[International Journal of Pharmaceutics Vol. 41:213-217(1988)]；化学交联微球的制备，如文献[Science Vol. 213:233-235(1981)]所报道，用戊二醛交联蛋白，然后洗涤并保存。Protein particles have also been reported by many literatures as carriers of pharmacologically active substances and diagnostic reagents. Among them, albumin microspheres can be prepared by thermal cross-linking and chemical cross-linking. Thermally crosslinked microspheres are prepared from emulsified mixtures (such as albumin, pharmacologically active substances to be entrapped and appropriate oil phases) at 100-150°C. The microspheres are then washed with an appropriate solvent and stored. Leucuta et al reported the preparation method of thermally crosslinked microspheres [International Journal of PharmaceuticsVol. 41:213-217(1988)]; preparation of chemically cross-linked microspheres, as reported in literature [Science Vol. 213:233-235(1981)], use glutaraldehyde to cross-link protein, then wash and preserve .
而且，现有技术中蛋白纳米粒的制备方法很难保持蛋白的生物活性。而且，难溶于水的药理活性物质也很难被包入蛋白纳米粒中，因为这种方法本身依靠乳化液的水相交联。水溶性药理活性物质因其溶于含蛋白的水相而能够被交联进入蛋白阵列，而水溶性差或脂溶性药理活性物质难以进入这种方法所形成的蛋白阵列中。Moreover, the preparation methods of protein nanoparticles in the prior art are difficult to maintain the biological activity of the protein. Moreover, it is also difficult for pharmacologically active substances that are poorly soluble in water to be incorporated into protein nanoparticles, because this method itself relies on the aqueous phase crosslinking of the emulsion. Water-soluble pharmacologically active substances can be cross-linked into the protein array because they are soluble in the protein-containing aqueous phase, while poorly water-soluble or fat-soluble pharmacologically active substances are difficult to enter into the protein array formed by this method.
发明内容Contents of the invention
本发明的技术目的在于提供一种制备用于体内递送药理活性物质的蛋白纳米粒的方法，以解决某些药理活性物质由于其疏水的性质而导致无法体内递送，以及形成纳米粒之后的蛋白质活性丧失的问题。The technical purpose of the present invention is to provide a method for preparing protein nanoparticles for in vivo delivery of pharmacologically active substances, to solve the problem that some pharmacologically active substances cannot be delivered in vivo due to their hydrophobic properties, and the protein activity after forming nanoparticles problem of loss.
为了实现本发明的技术目的，本发明的技术方案为：In order to realize the technical purpose of the present invention, technical scheme of the present invention is:
一种制备用于体内递送药理活性物质的蛋白纳米粒的方法，包括以下步骤：(a)用第一种溶剂溶解蛋白获得蛋白溶液；(b)在变性剂或者适合的变性条件中将药理活性物质加入步骤(a)中所述的蛋白溶液中，使蛋白展开与再折叠或自组装，把药理活性物质包入蛋白，形成蛋白纳米粒。A method for preparing protein nanoparticles for delivering pharmacologically active substances in vivo, comprising the following steps: (a) dissolving the protein with a first solvent to obtain a protein solution; (b) dissolving the pharmacologically active substance in a denaturing agent or suitable denaturing conditions Substances are added to the protein solution described in step (a), so that the protein is unfolded and refolded or self-assembled, and the pharmacologically active substance is encapsulated into the protein to form protein nanoparticles.
本发明技术方案中所述的蛋白纳米粒平均粒径为5～2000 nm，优化的为25～500 nm，最优的为50～300 nm。The average particle size of the protein nanoparticles described in the technical solution of the present invention is 5-2000 nm, preferably 25-500 nm, and most optimally 50-300 nm.
本发明技术方案中所述的蛋白纳米粒可包载占蛋白纳米粒总重量1%～40%的药理活性物质。The protein nanoparticles described in the technical solution of the present invention can carry pharmacologically active substances accounting for 1% to 40% of the total weight of the protein nanoparticles.
本发明技术方案所述的药理活性物质是疏水性药理活性物质，如抗肿瘤药物，心血管药物，抗炎药物，降糖药物，中枢神经系统药物，免疫抑制药物，以及抗病毒药物。The pharmacologically active substances described in the technical solution of the present invention are hydrophobic pharmacologically active substances, such as antineoplastic drugs, cardiovascular drugs, anti-inflammatory drugs, hypoglycemic drugs, central nervous system drugs, immunosuppressive drugs, and antiviral drugs.
其中，所述的疏水性药理活性物质是紫杉醇、多西紫杉醇、伊立替康、5-氟尿嘧啶、卡莫司汀、阿霉素、苯芥胆甾醇、哌泊舒凡、他莫昔芬、洛莫司汀、藤黄酸、冬凌草甲素、鬼臼毒素、阿托伐他汀、斯伐他汀、非诺贝特、硝苯地平、布洛芬、吲哚美辛、吡罗昔康、格列本脲、地西泮、利哌利酮、齐拉西酮、他克莫司、雷帕霉素、茚地那韦、利托那韦、Telaprevir（CAS号402957-28-2）、洛匹那韦、或它们的组合。Wherein, the hydrophobic pharmacologically active substance is paclitaxel, docetaxel, irinotecan, 5-fluorouracil, carmustine, doxorubicin, phenesin, pibosulfan, tamoxifen, Mustin, gambogic acid, oridonin, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, piroxicam, glucocorticoids Benurea, Diazepam, Risperidone, Ziprasidone, Tacrolimus, Rapamycin, Indinavir, Ritonavir, Telaprevir (CAS No. 402957-28-2), Lopi Navir, or a combination thereof.
其中，优选的疏水性药理活性物质是紫杉醇或多西紫杉醇。Among them, the preferred hydrophobic pharmacologically active substance is paclitaxel or docetaxel.
本发明技术方案所述的药理活性物质是亲水性药理活性物质。The pharmacologically active substance described in the technical solution of the present invention is a hydrophilic pharmacologically active substance.
其中，所述的亲水性药理活性物质是环磷酰胺、博莱霉素、道诺霉素、表柔比星、甲氨蝶呤、5-氟尿嘧啶或其类似物、铂或其类似物、长春碱或其类似物、高三尖杉酯碱或其衍生物、放线菌素-D、丝裂霉素-C、依托泊苷、或它们的组合。Wherein, the hydrophilic pharmacologically active substance is cyclophosphamide, bleomycin, daunomycin, epirubicin, methotrexate, 5-fluorouracil or its analogs, platinum or its analogs, Vinblastine or an analog thereof, homoharringtonine or a derivative thereof, actinomycin-D, mitomycin-C, etoposide, or combinations thereof.
本发明技术方案中所述的蛋白是白蛋白、转铁蛋白、胰岛素、血管内皮抑素、血红蛋白、肌红蛋白、溶菌酶、免疫球蛋白、α-2-巨球蛋白、纤维连接蛋白、纤层蛋白、胶原蛋白、明胶、人造多肽与蛋白，或者它们的组合。The protein described in the technical solution of the present invention is albumin, transferrin, insulin, vascular endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, fibronectin, Laminin, collagen, gelatin, artificial peptides and proteins, or combinations thereof.
其中，优选的蛋白是白蛋白、转铁蛋白、胰岛素、血管内皮抑素或血红蛋白。Among them, the preferred protein is albumin, transferrin, insulin, endostatin or hemoglobin.
本发明技术方案所述的步骤(a)的第一种溶剂是水，生理盐水，糖，冻干保护剂或蛋白稳定剂。其中，所述的冻干保护剂是磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，葡萄糖、或它们的组合。所述的蛋白稳定剂是海藻糖，甘露醇，蔗糖，乙酰色氨酸，辛酸钠或者它们的组合。The first solvent of step (a) described in the technical solution of the present invention is water, physiological saline, sugar, lyoprotectant or protein stabilizer. Wherein, the lyoprotectant is phosphate, acetate, glycine, tris, hydrogen peroxide, glutathione, glucose, or a combination thereof. The protein stabilizer is trehalose, mannitol, sucrose, acetyl tryptophan, sodium caprylate or their combination.
其中，所述的第一种溶剂优选水，磷酸盐，醋酸盐，乙酰色氨酸，辛酸钠或生理盐水。Wherein, the first solvent is preferably water, phosphate, acetate, acetyl tryptophan, sodium octanoate or physiological saline.
本发明技术方案中所述的步骤(a)的操作温度为-20～100℃；优选为50～85℃；最优为55～75℃。The operating temperature of step (a) described in the technical solution of the present invention is -20-100°C; preferably 50-85°C; optimally 55-75°C.
本发明技术方案中所述的步骤(a)的操作pH值为pH3～9；优选为pH5～8.5；最优为pH6～8。The operating pH value of the step (a) described in the technical solution of the present invention is pH 3-9; preferably pH 5-8.5; optimally pH 6-8.
本发明技术方案中所述的步骤(b)的变性剂或者适合的变性条件包括水、强酸、强碱、无机盐、有机溶剂、结构展开剂或表面活性剂。其中，所述的强酸强碱包括盐酸，硫酸，氢氧化钠等。所述的有机溶剂是甲醇，乙醇，异丙醇，福尔马林，氯仿，丙酮，硫化氢或它们的组合。所述的结构展开剂是水，2-巯基乙醇，二硫苏糖醇，盐酸胍，尿素，高氯酸，三丁基膦，甲巯丙脯氨酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸或它们的组合。所述的无机盐是水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，葡萄糖，蔗糖，甘露醇，海藻糖，乙酰色氨酸，辛酸钠或它们的组合。The denaturing agent or suitable denaturing conditions in step (b) described in the technical solution of the present invention include water, strong acid, strong base, inorganic salt, organic solvent, structure developing agent or surfactant. Wherein, the strong acid and strong base include hydrochloric acid, sulfuric acid, sodium hydroxide and the like. The organic solvent is methanol, ethanol, isopropanol, formalin, chloroform, acetone, hydrogen sulfide or their combination. Described structure developing agent is water, 2-mercaptoethanol, dithiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, captopril, performic acid, penicillamine, glutathione Glycerin, methimazole, acetylcysteine, or a combination thereof. The inorganic salts are water, sodium chloride, phosphate, acetate, glycine, tris, hydrogen peroxide, glutathione, glucose, sucrose, mannitol, trehalose, acetyl tryptophan acid, sodium caprylate or a combination thereof.
其中，优选的变性剂或者适合的变性条件是水，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，氯化钠，葡萄糖，乙醇，丙酮，硫化氢，2-巯基乙醇，尿素或它们的组合。Among them, the preferred denaturing agent or suitable denaturing conditions are water, phosphate, acetate, glycine, tris, sodium chloride, glucose, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea or their combination.
本发明技术方案中所述的变性剂或者适合的变性条件的pH值为pH3～9；优选为pH5.5～8.5。The pH value of the denaturant or suitable denaturing conditions described in the technical solution of the present invention is pH 3-9; preferably pH 5.5-8.5.
本发明技术方案中所述的步骤(b)还包括外力操作以辅助蛋白展开。Step (b) described in the technical solution of the present invention also includes external force operation to assist protein unfolding.
其中，所述的外力包括变化温度、变化压力、施加机械力或辐射。Wherein, the external force includes changing temperature, changing pressure, applying mechanical force or radiation.
其中，所述的变化压力为对反应施加10～100,000 psi的压力；优选的对反应施加2000～60,000 psi的压力。Wherein, the change pressure is to apply 10 to 100,000 pressure to the reactionPressure in psi; preferably a pressure of 2000 to 60,000 psi is applied to the reaction.
本发明的技术方案的方法进一步包括：(c)将蛋白纳米粒透析除去多余的小分子化合物或者进一步浓缩。The method of the technical solution of the present invention further includes: (c) dialyzing the protein nanoparticle to remove excess small molecule compounds or further concentrating.
本发明的技术方案的方法进一步包括：(d)将透析后的蛋白纳米粒经过脱水步骤制备成药物制剂。The method of the technical solution of the present invention further includes: (d) preparing the protein nanoparticle after dialysis into a pharmaceutical preparation through a dehydration step.
其中，所述的脱水的步骤是冷冻干燥、喷雾干燥或减压蒸馏。Wherein, the dehydration step is freeze-drying, spray-drying or vacuum distillation.
下面是对本发明技术方案的进一步描述：Below is a further description of the technical solution of the present invention:
除上述总的技术方案以外，本发明进一步提出了一种制备用于体内递送疏水性药理活性物质的纳米粒制备方法，所述方法包含以下几个步骤：(a)在-20～100℃，pH3～9的条件下，用第一种溶剂溶解所述蛋白获得蛋白溶液；(b)在变性剂或者适合的变性条件下，将疏水性药理活性物质加入步骤(a)中所述的蛋白溶液，从而引起蛋白展开与再折叠或自组装，把药理活性物质包入蛋白。形成的纳米粒平均粒径为5～500 nm，可包载占粒子总重量约1～40%的疏水性药理活性物质。这里的疏水性药理活性物质可以包括但不局限于紫杉醇、多西紫杉醇、伊立替康、卡莫司汀、阿霉素、苯芥胆甾醇、哌泊舒凡、他莫昔芬、洛莫司汀、藤黄酸、冬凌草甲素、鬼臼毒素、阿托伐他汀、斯伐他汀、非诺贝特、硝苯地平、布洛芬、吲哚美辛、吡罗昔康、格列本脲、地西泮、利哌利酮、齐拉西酮、他克莫司、雷帕霉素、茚地那韦、利托那韦、Telaprevir、洛匹那韦，以及它们的组合。In addition to the above-mentioned general technical scheme, the present invention further proposes a method for preparing nanoparticles for delivery of hydrophobic pharmacologically active substances in vivo. The method includes the following steps: (a) at -20 to 100°C, Under the condition of pH 3-9, dissolving the protein with the first solvent to obtain a protein solution; (b) adding a hydrophobic pharmacologically active substance to the protein solution described in step (a) under a denaturing agent or suitable denaturing conditions , thereby causing protein unfolding and refolding or self-assembly, and encapsulating pharmacologically active substances into proteins. The formed nanoparticles have an average particle diameter of 5-500 nm, and can carry hydrophobic pharmacologically active substances accounting for about 1-40% of the total weight of the particles. The hydrophobic pharmacologically active substances here may include, but are not limited to, paclitaxel, docetaxel, irinotecan, carmustine, doxorubicin, phenerine, piposulfan, tamoxifen, lolimus Tin, gambogic acid, oridonin, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, piroxicam, glibenclamide , diazepam, risperidone, ziprasidone, tacrolimus, rapamycin, indinavir, ritonavir, Telaprevir, lopinavir, and combinations thereof.
本技术方案的方法中的蛋白一般可以包括但不局限于白蛋白、转铁蛋白、胰岛素、内皮抑素、血红蛋白、肌红蛋白、溶菌酶、免疫球蛋白、α-2-巨球蛋白、纤维连接蛋白、纤层蛋白、胶原蛋白、明胶、人造肽与蛋白，以及它们的组合。本实施方案的方法中的第一种溶剂可以包括水、生理盐水、磷酸盐、醋酸盐、甘氨酸、三羟甲基氨基甲烷、过氧化氢、谷胱甘肽、葡萄糖、海藻糖、甘露醇、蔗糖、乙酰色氨酸、辛酸钠，以及它们的组合。此外，本实施方案的方法中的变性剂或者适合的变性条件可以包括水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，甲醇，乙醇，异丙醇，福尔马林，氯仿，丙酮，硫化氢，2-巯基乙醇，二硫苏糖醇，盐酸胍，尿素，高氯酸，三丁基膦，甲巯丙脯氨酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸以及它们的组合。Proteins in the method of this technical solution generally include but are not limited to albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fiber Connexin, lamin, collagen, gelatin, artificial peptides and proteins, and combinations thereof. The first solvent in the method of this embodiment may include water, physiological saline, phosphate, acetate, glycine, tris, hydrogen peroxide, glutathione, glucose, trehalose, mannitol , sucrose, acetyl tryptophan, sodium caprylate, and combinations thereof. In addition, the denaturant or suitable denaturing conditions in the method of this embodiment may include water, sodium chloride, phosphate, acetate, glycine, tris, hydrogen peroxide, glutathione, methanol , ethanol, isopropanol, formalin, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, captopril , performic acid, penicillamine, glutathione, methimazole, acetylcysteine, and combinations thereof.
其中，所述的纳米粒子的平均直径优选为25 nm～500 nm；最优为50 nm～300 nm。所述步骤(a)优选在50℃～85℃进行，最优在55℃～75℃进行。pH优选在5～8.5的条件下进行，最优在6～8的条件下进行。Wherein, the average diameter of the nanoparticles is preferably 25 nm-500 nm; optimally 50 nm-300 nm. The step (a) is preferably carried out at 50°C to 85°C, most preferably at 55°C to 75°C. The pH is preferably carried out under the condition of 5-8.5, and most preferably carried out under the condition of 6-8.
所述变性剂或者适合的变性条件是水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，乙醇，丙酮，硫化氢，2-巯基乙醇，尿素或它们的组合。The denaturant or suitable denaturing conditions are water, sodium chloride, phosphate, acetate, glycine, tris, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea or combinations thereof.
其中，所述的变性剂或者适合的变性条件的pH值为3～9，优选为5.5～8.5。Wherein, the pH value of the denaturant or suitable denaturing conditions is 3-9, preferably 5.5-8.5.
上述方法进一步包括：(c)将纳米粒透析除去多余的小分子化合物或者进一步浓缩。还进一步包括：(d) 将透析后的纳米粒经过脱水步骤制备成药物制剂。其中，所述的脱水步骤是冻干，减压蒸馏或喷雾干燥。The above method further includes: (c) dialyzing the nanoparticles to remove excess small molecule compounds or further concentrating them. It further includes: (d) preparing the dialyzed nanoparticles into a pharmaceutical preparation through a dehydration step. Wherein, the dehydration step is lyophilization, vacuum distillation or spray drying.
本发明技术方案再进一步提出了一种制备用于体内递送紫杉醇蛋白纳米粒的方法，所述方法包含以下几个步骤：(a)在55～75℃，pH6～8的条件下，用第一种溶剂溶解所述蛋白获得蛋白溶液；(b)在变性剂或者适合的变性条件下，将紫杉醇加入步骤(a)中所述的蛋白溶液，从而引起蛋白展开与再折叠或自组装，把紫杉醇包在所述蛋白中；(c)将纳米粒透析除去多余的小分子化合物或者进一步浓缩；(d)对所得溶液进行脱水作用，制得能够保存的药物剂型。这里制得的纳米粒平均粒径为50～300 nm，可包载占粒子总重量约1～40%的药理活性物质。本实施方案的方法中的变性剂或者适合的变性条件可以从水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，甲醇，乙醇，异丙醇，福尔马林，氯仿，丙酮，硫化氢，2-巯基乙醇，二硫苏糖醇，盐酸胍，尿素，高氯酸，三丁基膦，甲巯丙脯氨酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸以及它们的组合中选择。此外，本实施方案的方法中的蛋白可以从白蛋白、转铁蛋白、胰岛素、内皮抑素、血红蛋白中选择。The technical solution of the present invention further proposes a method for preparing paclitaxel protein nanoparticles for in vivo delivery. A solvent dissolves the protein to obtain a protein solution; (b) under a denaturing agent or suitable denaturing conditions, paclitaxel is added to the protein solution described in step (a), thereby causing the protein to unfold and refold or self-assemble, and the paclitaxel encapsulation in the protein; (c) dialyzing the nanoparticles to remove excess small molecule compounds or further concentrating; (d) dehydrating the obtained solution to prepare a pharmaceutical dosage form that can be preserved. The nanoparticles prepared here have an average particle size of 50-300 nm, and can contain pharmacologically active substances accounting for about 1-40% of the total weight of the particles. The denaturant or suitable denaturing conditions in the method of the present embodiment can be selected from water, sodium chloride, phosphate, acetate, glycine, trishydroxymethyl aminomethane, hydrogen peroxide, glutathione, methanol, ethanol , isopropanol, formalin, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, captopril, over Formic acid, penicillamine, glutathione, methimazole, acetylcysteine, and combinations thereof. In addition, the protein in the method of this embodiment can be selected from albumin, transferrin, insulin, endostatin, and hemoglobin.
本发明技术方案再进一步提出了一种将多西紫杉醇包入蛋白用于体内递送的纳米粒制备方法，这种方法包含以下几个步骤：(a)在55～75℃，pH6～8的条件下用第一种溶液溶解所述蛋白获得蛋白溶液；(b)在变性剂或者适合的变性条件下，将多西紫杉醇加入到步骤(a)中所述的蛋白溶液，从而引起展开与再折叠或自组装，把多西紫杉醇包入蛋白；(c) 将纳米粒透析除去多余的小分子化合物或者进一步浓缩；(d) 将透析后的纳米粒经过脱水步骤制备成药物制剂；这里制得的纳米粒平均粒径为约50～300 nm，可包载占粒子总重量约1～40%的多烯紫杉醇。本实施方案的方法中的变性剂或者适合的变性条件可以从水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，甲醇，乙醇，异丙醇，福尔马林，氯仿，丙酮，硫化氢，2-巯基乙醇，二硫苏糖醇，盐酸胍，尿素，高氯酸，三丁基膦，甲巯丙脯氨酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸以及它们的组合中选择。此外，本实施方案的方法中的蛋白可以从白蛋白、转铁蛋白、胰岛素、内皮抑素、血红蛋白中选择。The technical solution of the present invention further proposes a method for preparing nanoparticles that encapsulate docetaxel into protein for in vivo delivery. This method includes the following steps: (a) at 55-75°C, pH 6-8 Dissolving the protein with the first solution to obtain a protein solution; (b) adding docetaxel to the protein solution described in step (a) under a denaturing agent or suitable denaturing conditions, thereby causing unfolding and refolding or self-assembly, including docetaxel in proteins; (c) dialysis of nanoparticles to remove excess small molecule compounds or further concentration; (d) preparation of pharmaceutical preparations through dehydration steps of nanoparticles after dialysis; the prepared here The average particle diameter of the nanoparticles is about 50-300 nm, and can contain docetaxel accounting for about 1-40% of the total weight of the particles. The denaturant or suitable denaturing conditions in the method of the present embodiment can be selected from water, sodium chloride, phosphate, acetate, glycine, trishydroxymethyl aminomethane, hydrogen peroxide, glutathione, methanol, ethanol , isopropanol, formalin, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, captopril, over Formic acid, penicillamine, glutathione, methimazole, acetylcysteine, and combinations thereof. In addition, the protein in the method of this embodiment can be selected from albumin, transferrin, insulin, endostatin, and hemoglobin.
本发明技术方案再进一步提出了一种蛋白包载药理活性物质用于体内递送的纳米粒，制备这种纳米粒的方法包含以下几个步骤：(a)在-20～100℃，pH3～9的条件下，用第一种溶剂溶解所述蛋白获得蛋白溶液；(b) 在变性剂或者适合的变性条件下，将药理活性物质加入到步骤(a)所述的蛋白溶液中，使蛋白展开与再折叠或自组装，将药物包裹在所述蛋白中。The technical solution of the present invention further proposes a nanoparticle for in vivo delivery of a protein-loaded pharmacologically active substance. The method for preparing the nanoparticle includes the following steps: (a) at -20-100°C, pH3-9 Under the conditions, the first solvent is used to dissolve the protein to obtain a protein solution; (b) under a denaturing agent or suitable denaturing conditions, the pharmacologically active substance is added to the protein solution described in step (a) to expand the protein As with refolding or self-assembly, the drug is encapsulated within the protein.
本发明技术方案再进一步提出了一种蛋白包载紫杉醇用于体内递送的纳米粒，制备这种纳米粒的方法包含以下几个步骤：(a)在55～75℃，pH6～8的条件下，用第一种溶剂溶解所述蛋白获得蛋白溶液；(b) 在变性剂或者适合的变性条件下，将紫杉醇加入到步骤(a)所述的蛋白溶液中，使蛋白展开与再折叠或自组装，将药物包裹在所述蛋白中；(c)透析形成的纳米粒溶液以去除过剩的物质，得到高浓度的溶液；(d)对所得溶液进行脱水作用，制得能够保存的药品剂型。The technical solution of the present invention further proposes a protein-loaded paclitaxel nanoparticle for in vivo delivery. The method for preparing this nanoparticle includes the following steps: (a) at 55-75°C and pH6-8 , using the first solvent to dissolve the protein to obtain a protein solution; (b) adding paclitaxel to the protein solution described in step (a) under a denaturing agent or suitable denaturing conditions, so that the protein unfolds and refolds or automatically Assembling, wrapping the drug in the protein; (c) dialyzing the formed nanoparticle solution to remove excess substances to obtain a high-concentration solution; (d) dehydrating the obtained solution to prepare a pharmaceutical dosage form that can be preserved.
本发明技术方案再进一步提出了一种蛋白包载多西紫杉醇用于体内递送的纳米粒，制备这种纳米粒的方法包含以下几个步骤：(a)在55～75℃，pH6～8的条件下，用第一种溶剂溶解所述蛋白获得蛋白溶液；(b) 在变性剂或者适合的变性条件下，将多西紫杉醇加入到步骤(a)所述的蛋白溶液中，使蛋白展开与再折叠或自组装，将药物包裹在所述蛋白中；(c)透析形成的纳米粒溶液以去除过剩的物质，得到高浓度的溶液；(d)对所得溶液进行脱水作用，制得能够保存的药品剂型。The technical scheme of the present invention further proposes a protein-loaded docetaxel nanoparticle for in vivo delivery. The method for preparing this nanoparticle includes the following steps: Under the following conditions, the protein is dissolved with the first solvent to obtain a protein solution; (b) under denaturing agent or suitable denaturing conditions, docetaxel is added to the protein solution described in step (a), so that the protein unfolds and Refolding or self-assembly encapsulates the drug in the protein; (c) dialyzing the formed nanoparticle solution to remove excess material to obtain a high-concentration solution; (d) dehydrating the resulting solution to produce a pharmaceutical dosage forms.
对本发明所述技术方案的说明：Description of the technical solution of the present invention:
本发明所述的“纳米粒”，指的是小的单位，而在转运和性质方面特指为一个整体。本发明所制备的蛋白纳米粒平均粒径分布于5 nm到2000 nm之间。一个更好的区间是25 nm到500 nm，再一个更好的区间是50 nm到300 nm。而且，本发明所制备的纳米粒能够结合高达40%的药理活性物质。The "nanoparticle" mentioned in the present invention refers to a small unit, and specifically refers to a whole in terms of transport and properties. The average particle size distribution of the protein nanoparticles prepared by the invention is between 5 nm and 2000 nm. A better range is 25 nm to 500 nm, and a better range is 50 nm to 300 nm. Moreover, the nanoparticles prepared in the present invention can bind up to 40% of pharmacologically active substances.
本发明中所述的将药理活性物质包入蛋白，指的是药理活性物质可以通过蛋白的展开和再折叠，进入蛋白中心区域。一般来说，药理活性物质包括在：给病人服用时，能够产生药理反应的任何的物质。本发明中的药理活性物质包括疏水和亲水的化合物。本领域技术人员清楚疏水性药理活性物质的水溶性不好，亲水性药理活性物质能够优化的溶解在水中。疏水性的药理活性物质包括如下的化合物，但是不仅仅限于此：抗肿瘤药物，心血管药物，抗炎药物，降糖药物，中枢神经系统药物，免疫抑制药物，以及抗病毒药物。The encapsulation of the pharmacologically active substance in the protein in the present invention means that the pharmacologically active substance can enter the central region of the protein through unfolding and refolding of the protein. In general, pharmacologically active substances include: any substance that can produce a pharmacological response when administered to a patient. Pharmacologically active substances in the present invention include both hydrophobic and hydrophilic compounds. Those skilled in the art know that hydrophobic pharmacologically active substances have poor water solubility, and hydrophilic pharmacologically active substances can be optimally dissolved in water. Hydrophobic pharmacologically active substances include, but are not limited to, anticancer drugs, cardiovascular drugs, anti-inflammatory drugs, hypoglycemic drugs, central nervous system drugs, immunosuppressive drugs, and antiviral drugs.
本发明涉及的疏水性药理活性物质可以包括，但是不仅仅局限于此：紫杉醇、多西紫杉醇、伊立替康、卡莫司汀、阿霉素、苯芥胆甾醇、哌泊舒凡、他莫昔芬、洛莫司汀、藤黄酸、冬凌草甲素、鬼臼毒素、阿托伐他汀、斯伐他汀、非诺贝特、硝苯地平、布洛芬、吲哚美辛、吡罗昔康、格列本脲、地西泮、利哌利酮、齐拉西酮、他克莫司、雷帕霉素、茚地那韦、利托那韦、Telaprevir、洛匹那韦，以及它们的组合。The hydrophobic pharmacologically active substances involved in the present invention may include, but are not limited to: paclitaxel, docetaxel, irinotecan, carmustine, doxorubicin, phenerethinate, pibosufan, tamoxol Xifen, lomustine, gambogic acid, oridonin, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, pyridoxine Roxicam, glibenclamide, diazepam, risperidone, ziprasidone, tacrolimus, rapamycin, indinavir, ritonavir, telaprevir, lopinavir, and their The combination.
更优化的说, 具有药理活性的疏水性物质包括：抗肿瘤药理活性物质如紫杉醇，多西紫杉醇，伊立替康，卡氯芥，阿霉素，胆甾醇对苯乙酸氮芥，哌泊舒凡，他莫昔芬，环己亚硝脲，藤黄酸，冬凌草素甲，鬼臼毒素以及它们的衍生物，以及它们的组合。More optimally, hydrophobic substances with pharmacological activity include: antitumor pharmacologically active substances such as paclitaxel, docetaxel, irinotecan, carmustine, doxorubicin, cholesteryl phenylacetate, piposulfan , tamoxifen, cyclohexylnitrosourea, gambogic acid, oridonin A, podophyllotoxin and their derivatives, and their combinations.
在一个更优化的范围中，疏水性药理活性物质包括紫杉醇和多西紫杉醇。上述的药理活性物质包括晶体形式和无定形形式，其晶体形式包括带有结晶水和不带有结晶水的形式。In a more preferred range, hydrophobic pharmacologically active substances include paclitaxel and docetaxel. The aforementioned pharmacologically active substances include crystalline forms and amorphous forms, and the crystalline forms include forms with and without water of crystallization.
本发明所涉及的药理活性物质还包括亲水性物质。The pharmacologically active substances involved in the present invention also include hydrophilic substances.
亲水性物质可以包括如下化合物，但是不仅仅局限于此：环磷酰胺、博莱霉素、道诺霉素、表柔比星、甲氨蝶呤、5-氟尿嘧啶及其类似物、铂及其类似物、长春碱及其类似物、高三尖杉酯碱及其衍生物、放线菌素-D，丝裂霉素-C，依托泊苷以及它们的组合。Hydrophilic substances may include, but are not limited to, the following compounds: cyclophosphamide, bleomycin, daunomycin, epirubicin, methotrexate, 5-fluorouracil and its analogs, platinum and Analogs thereof, vinblastine and its analogs, homoharringtonine and its derivatives, actinomycin-D, mitomycin-C, etoposide and combinations thereof.
本领域技术人员能够理解本发明中使用的药理活性物质的量会根据蛋白的量的变化而变化，同时根据纳米粒的量的变化而变化。同时，有经验的人能够意识到本发明中使用的药理活性物质，可以是纯的物质，或者是混合物，这些都没有背离本发明的范围。Those skilled in the art can understand that the amount of the pharmacologically active substance used in the present invention will vary according to the amount of protein, and will also vary according to the amount of nanoparticles. At the same time, those skilled in the art can realize that the pharmacologically active substances used in the present invention can be pure substances or mixtures without departing from the scope of the present invention.
本发明中，可以选择不同的蛋白去形成本领域技术人员所感兴趣的纳米粒子。本发明中所涉及的蛋白包括所有的能够展开并结合药理活性物质的蛋白或者多肽。合适的蛋白的例子包括如下，但是不仅仅局限于此：白蛋白，转铁蛋白，胰岛素，血管内皮抑素，血红蛋白，肌球蛋白，溶菌酶，免疫球蛋白，α-2-巨球蛋白，纤连蛋白，核纤层蛋白，胶原蛋白，明胶，人造蛋白以及它们的组合。In the present invention, different proteins can be selected to form nanoparticles of interest to those skilled in the art. The proteins involved in the present invention include all proteins or polypeptides that can unfold and bind pharmacologically active substances. Examples of suitable proteins include, but are not limited to, albumin, transferrin, insulin, endostatin, hemoglobin, myosin, lysozyme, immunoglobulin, alpha-2-macroglobulin, Fibronectin, lamin, collagen, gelatin, artificial proteins and combinations thereof.
在一个更优化的范围，适合于本发明的蛋白包括如下：白蛋白，转铁蛋白，胰岛素，血管内皮抑素，血红蛋白以及它们的组合物。本领域技术人员会清楚本发明方法中所使用的蛋白的量随着活性物质的量和纳米粒的量的变化而变化[Annalytical Biochemistry Vol. 72:248-254(1976)].In a more optimal range, proteins suitable for the present invention include the following: albumin, transferrin, insulin, endostatin, hemoglobin and their combinations. It will be clear to those skilled in the art that the amount of protein used in the methods of the invention varies with the amount of active substance and the amount of nanoparticles [AnnalyticalBiochemistry Vol. 72:248-254(1976)].
本发明方法中的步骤(a)为用第一种溶剂溶解所述蛋白获得蛋白溶液。这里的蛋白溶液指的是溶液中包括蛋白和能够溶解蛋白的溶剂，在蛋白展开之后，能够再折叠或者自组装。这里使用的再折叠指的是一个解折叠或者变性的蛋白能够再折叠恢复到合适的三维结构的过程。这里使用的自组装指的是再折叠的蛋白结合到一起形成纳米粒的过程。本领域技术人员清楚蛋白的再折叠过程可以通过很多条件完成。在蛋白溶液中使用的第一种溶液举例如下，但不仅仅局限于此：水，生理盐水，糖，冻干保护剂和蛋白稳定剂，在更精确的范围，溶剂包括水，氯化钠溶液，磷酸盐溶液，醋酸溶液，甘氨酸溶液，三羟甲基氨基甲烷溶液，过氧化氢水溶液，谷胱甘肽水溶液，葡萄糖溶液，海藻糖溶液，甘露醇溶液，蔗糖溶液，乙酰色氨酸溶液，辛酸钠溶液，以及它们的混合物。Step (a) in the method of the present invention is to dissolve the protein with the first solvent to obtain a protein solution. The protein solution here refers to a solution that includes a protein and a solvent capable of dissolving the protein, and after the protein unfolds, it can refold or self-assemble. Refolding as used herein refers to the process by which an unfolded or denatured protein can be refolded back into a proper three-dimensional structure. Self-assembly as used here refers to the process by which refolded proteins join together to form nanoparticles. It is clear to those skilled in the art that the refolding process of proteins can be accomplished through many conditions. Examples of first solutions used in protein solutions include, but are not limited to: water, saline, sugar, lyoprotectants and protein stabilizers, and to a more precise extent, solvents include water, sodium chloride solution , Phosphate Solution, Acetic Acid Solution, Glycine Solution, Tris Hydroxymethylaminomethane Solution, Hydrogen Peroxide Solution, Glutathione Solution, Glucose Solution, Trehalose Solution, Mannitol Solution, Sucrose Solution, Acetyl Tryptophan Solution, Sodium octanoate solutions, and mixtures thereof.
在一个更加精确的范围，蛋白溶液中的溶剂包括水，磷酸盐，醋酸盐和氯化钠溶液。本发明中所使用的蛋白溶液中的溶剂的浓度只要适合溶解蛋白和蛋白再折叠，都是可行的。总体上来说，蛋白溶液中溶剂的含量范围从0.001 M到1.6 M。优化的范围，从0.03 M到1.5 M。再优化的范围，从0.05 M到0.8 M。一个更加优化的范围，从0.1 M到0.3 M。本领域技术人员能够明白用来溶解蛋白的溶剂的量会根据蛋白的量和蛋白溶液浓度的变化而变化。On a more precise scale, solvents in protein solutions include water, phosphate, acetate and sodium chloride solutions. The concentration of the solvent in the protein solution used in the present invention is feasible as long as it is suitable for protein dissolution and protein refolding. Generally speaking, the content of solvent in protein solution ranges from 0.001M to 1.6 M. Optimized range from 0.03M to 1.5 M. Re-optimized range from 0.05M to 0.8 M. A more optimized range, from 0.1 M to 0.3 M. Those skilled in the art can understand that the amount of solvent used to dissolve the protein will vary according to the amount of protein and the concentration of the protein solution.
实验表明，本发明中的步骤(a)中的反应参数对于形成纳米粒来说，是非常重要的。一般来说，要获得一个理想的结果，本发明中的步骤(a)须在-20℃到100℃范围之间反应。一个比较精确的范围是从50℃到85℃。一个更加精确的范围是从55℃到75℃。实验已经证明，要获得一个比较理想的结果，本发明中步骤(a)的pH须在3到9之间，一个比较精确的范围是从5到8.5，在一个更加精确的范围是从6到8。本领域技术人员能够清楚步骤(a)需要一段时间以使得蛋白充分的溶解。一般来说，时间的长短依赖于所使用的蛋白的种类，蛋白的使用量，使用溶剂的种类，溶剂的含量，溶剂的浓度和其他的一些因素。一般来说，本领域技术人员能够充分意识到，反应过程和反应过程的每一个步骤都需要充足的时间，举一个例子，反应过程需要5分钟到8小时不等。Experiments show that the reaction parameters in the step (a) of the present invention are very important for the formation of nanoparticles. Generally speaking, to obtain a desired result, the step (a) in the present invention must be reacted in the range of -20°C to 100°C. A more precise range is from 50°C to 85°C. A more precise range is from 55°C to 75°C. Experiments have proved that to obtain a more ideal result, the pH of step (a) must be between 3 and 9 among the present invention, and a more accurate range is from 5 to 8.5, and is from 6 to 8.5 in a more accurate range. 8. Those skilled in the art can clearly understand that step (a) requires a period of time to fully dissolve the protein. Generally speaking, the length of time depends on the type of protein used, the amount of protein used, the type of solvent used, the content of solvent, the concentration of solvent and other factors. Generally speaking, those skilled in the art can fully realize that the reaction process and each step of the reaction process require sufficient time. For example, the reaction process takes 5 minutes to 8 hours.
本发明的第二个步骤即步骤(b)包括在变性剂或者适合的变性条件下，将药理活性物质加入到经过步骤(a)所述的蛋白溶液中，使蛋白展开与再折叠或者自组装。这里使用的变性剂或者适合的变性条件指的是能诱导蛋白或者多肽改变它们的三维或者二维结构的溶液。一般来说，这里提到的变性剂或者适合的变性条件能够诱导蛋白温和的变性。本领域技术人员能够充分意识到温和变性指的是在蛋白解折叠/变性之后，能够在某些条件下（如使用再折叠溶液）再折叠成合适的结构。变性剂或者适合的变性条件可以提供一个环境来破坏蛋白的二硫键，形成氢键，以使得水能够干扰蛋白内部的疏水作用。本领域技术人员能够充分意识到许多溶液能够充当变性剂或者适合的变性条件。 这里所述的变性剂或者适合的变性条件括水，强酸，强碱，无机盐，有机溶剂，结构展开剂和表面活性剂。合适的变性剂或者适变性条件举例如下，但是不仅仅局限于此：水，氢氧化钠，盐酸，硫酸，甲醇溶液，乙醇溶液，异丙醇，福尔马林，氯仿，丙酮，硫化氢，2-巯基乙醇，二硫苏糖醇，胍 ,尿素，高氯酸，三正丁基膦, 甲巯丙脯酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸，氯化钠溶液，磷酸盐溶液，醋酸盐溶液，甘氨酸溶液，三羟甲基氨基甲烷溶液，过氧化氢，谷胱甘肽，葡萄糖，蔗糖，甘露醇，海藻糖，乙酰色氨酸，辛酸钠以及它们的混合物。在一个更精确的范围，变性剂或者适合的变性条件包括水，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，乙醇，丙酮，硫化氢，2-巯基乙醇，尿素。实验证明，当变性剂或者适合的变性条件的pH在3～9之间，获得的结果是令人满意的。一个更加精确的pH范围是从5.5到8.5。The second step of the present invention, that is, step (b), includes adding pharmacologically active substances to the protein solution described in step (a) under a denaturing agent or suitable denaturing conditions, so that the protein unfolds and refolds or self-assembles . The denaturing agent or suitable denaturing conditions used here refer to solutions that can induce proteins or polypeptides to change their three-dimensional or two-dimensional structures. In general, the denaturants or suitable denaturing conditions mentioned here can induce mild denaturation of proteins. Those skilled in the art are well aware that mild denaturation refers to the ability to refold into a proper structure under certain conditions (such as using a refolding solution) after the protein is unfolded/denatured. Denaturing agents or suitable denaturing conditions can provide an environment to break the disulfide bonds of proteins and form hydrogen bonds, so that water can interfere with the internal hydrophobic interactions of proteins. Those skilled in the art are well aware that many solutions can serve as denaturing agents or suitable denaturing conditions. The denaturing agent or suitable denaturing conditions described here include water, strong acid, strong base, inorganic salt, organic solvent, structure developing agent and surfactant. Suitable denaturants or suitable denaturing conditions are exemplified as follows, but not limited thereto: water, sodium hydroxide, hydrochloric acid, sulfuric acid, methanol solution, ethanol solution, isopropanol, formalin, chloroform, acetone, hydrogen sulfide, 2-mercaptoethanol, dithiothreitol, guanidine, urea, perchloric acid, tri-n-butylphosphine, captopril, performic acid, penicillamine, glutathione, methimazole, acetylcysteine Acidic acid, sodium chloride solution, phosphate solution, acetate solution, glycine solution, tris solution, hydrogen peroxide, glutathione, glucose, sucrose, mannitol, trehalose, acetyl tryptophan acid, sodium caprylate and mixtures thereof. On a more precise scale, denaturants or suitable denaturing conditions include water, phosphate, acetate, glycine, tris, ethanol, acetone, hydrogen sulfide, 2-mercaptoethanol, urea. Experiments have proved that when the pH of the denaturant or suitable denaturing conditions is between 3 and 9, the obtained results are satisfactory. A more precise pH range is from 5.5 to 8.5.
另外，除了变性剂，本发明中蛋白解折叠还可以使用外加压力。一般来说，外加压力包括能够致蛋白解折叠的力量。外加力包括：温度，压力变化，机械力，辐射。外加力包括从10到100,000 psi的压力，一个更合适的范围是从2000到60000 psi。In addition, in addition to denaturants, external pressure can also be used for protein unfolding in the present invention. In general, applied pressure includes forces capable of causing protein unfolding. Applied forces include: temperature, pressure change, mechanical force, radiation. Applied forces include pressures from 10 to 100,000 psi, with a more suitable range being from 2,000 to 60,000 psi.
本发明可能另外需要一个步骤(c)将纳米粒子透析除去多余的小分子化合物或者进一步浓缩。一般来说,这个步骤包括任意的能够将小分子从纳米粒中分离出去的方法，本领域技术人员能够充分意识到分离的方法包括任意的能够纯化蛋白或者多肽的方法。这些方法可包括盐沉淀，透析，层析以及它们的组合，这些方法可以适当的选择。一个更加精细的范围，透析是可行的。The present invention may additionally require a step (c) to dialyze the nanoparticles to remove excess small molecule compounds or further concentrate them. Generally speaking, this step includes any method capable of separating small molecules from nanoparticles, and those skilled in the art can fully appreciate that the method of separation includes any method capable of purifying proteins or polypeptides. These methods may include salt precipitation, dialysis, chromatography and combinations thereof, and these methods may be appropriately selected. On a finer scale, dialysis is available.
本发明可能还需要一个步骤(d) 将透析后的纳米粒经过脱水步骤制备成药物制剂。一般来说，保护的方法包括给纳米粒脱水以方便储存和运输，以致获得合适的剂型。本发明所涉及的保护的方法包括：离心，减压干燥，冻干，喷雾干燥。The present invention may also require a step (d) of dehydrating the dialyzed nanoparticles to prepare a pharmaceutical preparation. In general, methods of protection include dehydration of nanoparticles to facilitate storage and transportation, so as to obtain suitable dosage forms. The protection method involved in the present invention includes: centrifugation, drying under reduced pressure, freeze-drying and spray-drying.
本领域技术人员能够意识到本发明的范围和精髓是变动的。解折叠的物质是变化的，同时许多的药理活性物质是可使用的，许多的蛋白和合成的多肽也是可以用来作为载体的。本发明将会在下面的实施例中得到更加明确和清晰地描述。Those skilled in the art can appreciate that the scope and spirit of the present invention vary. The unfolded substances are variable, and many pharmacologically active substances are available, and many proteins and synthetic polypeptides can also be used as carriers. The present invention will be more clearly and clearly described in the following examples.
本发明的有益效果在于：The beneficial effects of the present invention are:
首先，经本发明提供的方法形成的纳米粒子可以达到90%的包封率，超过现有技术，形成了一种高效低耗的方法；其次，本发明所提供的方法能够获得最高达40%的载药量，即纳米粒中含有40%的药理活性物质。由于有更高的载药量，在治疗时，可以获得更小的用药体积，更短的用药时间，对病人更方便。较高的载药量降低了递送药理活性物质时蛋白的使用量，提高了产品的成本效率；最后，现有技术提供的较低载药能力不能满足高剂量给药，因为高剂量的给药需要很大的给药体积。然而，本实验发明的具有高载药能力的纳米粒不会受给药体积的限制。Firstly, the nanoparticles formed by the method provided by the present invention can achieve an encapsulation efficiency of 90%, surpassing the prior art, forming a method with high efficiency and low consumption; secondly, the method provided by the present invention can obtain up to 40% The drug loading capacity, that is, the nanoparticles contain 40% of pharmacologically active substances. Due to the higher drug loading, a smaller dosage volume and shorter medication time can be obtained during treatment, which is more convenient for patients. Higher drug loading reduces the amount of protein used to deliver pharmacologically active substances and increases the cost-effectiveness of the product; finally, the lower drug-loading capacity provided by existing technologies cannot meet high-dose administration, because high-dose administration Large dosing volumes are required. However, the nanoparticles with high drug loading capacity invented in this experiment will not be limited by the volume of administration.
通过本发明制备的纳米粒的另一个有益效果是，纳米粒能够特异性的递送药理活性物质。通过本发明制备的纳米粒子能够靶向一些身体器官和系统。比如，当用白蛋白作为载体时，通过改变纳米粒子的大小，靶向肝脏，肺，脾脏或者淋巴系统等。当用转铁蛋白或者胰岛素作为载体的时候，它们的受体在细胞表面高表达，通过载体和肿瘤细胞表面的亲和性，可以特异性的将药理活性物质输送到肿瘤组织。当载体是血管内皮抑素的时候，它的受体分布于血管细胞，由于肿瘤组织中存在大量的血管的缘故，纳米粒子能够大量的在肿瘤组织中蓄积。总之，不同的蛋白载体可以靶向不同的组织或者器官。因此可以说，本发明提供了一种高效低耗的方法来输送药理活性物质到身体的不同部位。Another beneficial effect of the nanoparticles prepared by the present invention is that the nanoparticles can specifically deliver pharmacologically active substances. Nanoparticles prepared by the present invention are capable of targeting several bodily organs and systems. For example, when albumin is used as a carrier, by changing the size of the nanoparticles, targeting the liver, lung, spleen or lymphatic system, etc. When transferrin or insulin is used as a carrier, their receptors are highly expressed on the cell surface, and the pharmacologically active substance can be specifically delivered to the tumor tissue through the affinity between the carrier and the surface of tumor cells. When the carrier is endostatin, its receptors are distributed in vascular cells, and because of the large number of blood vessels in the tumor tissue, the nanoparticles can accumulate in a large amount in the tumor tissue. In conclusion, different protein carriers can target different tissues or organs. Therefore, it can be said that the present invention provides an efficient and low-cost method to deliver pharmacologically active substances to different parts of the body.
附图说明Description of drawings
图1为不同pH对本发明中纳米粒粒径大小的影响（15%投药，药物/蛋白）Fig. 1 is the impact of different pH on the particle size of nanoparticles in the present invention (15% administration, drug/protein)
图2为本发明中白蛋白-紫杉醇纳米粒的粒径分布图Fig. 2 is the particle size distribution figure of albumin-paclitaxel nanoparticles in the present invention
图3为本发明中白蛋白-紫杉醇纳米粒的透射电子显微镜图像（载药量10.59%）Figure 3 is a transmission electron microscope image of albumin-paclitaxel nanoparticles in the present invention (drug loading 10.59%)
图4为紫杉醇-白蛋白纳米粒的高分辨率TEM 照片Figure 4 is a high-resolution TEM photo of paclitaxel-albumin nanoparticles
其中，(a)白蛋白-紫杉醇纳米粒；(b)放大的空载的白蛋白纳米粒；(c)放大的游离的紫杉醇；(d)放大的白蛋白-紫杉醇纳米粒Among them, (a) albumin-paclitaxel nanoparticles; (b) enlarged empty albumin nanoparticles; (c) enlarged free paclitaxel; (d) enlarged albumin-paclitaxel nanoparticles
图5为白蛋白-紫杉醇纳米粒的X射线粉末衍射图像Figure 5 is an X-ray powder diffraction image of albumin-paclitaxel nanoparticles
其中，(a)紫杉醇；(b)空白的白蛋白纳米粒；(c)紫杉醇-白蛋白纳米粒（载药量12.9%）；(d)白蛋白和紫杉醇的物理混合物(12.9%)Among them, (a) paclitaxel; (b) blank albumin nanoparticles; (c) paclitaxel-albumin nanoparticles (drug loading 12.9%); (d) physical mixture of albumin and paclitaxel (12.9%)
图6为白蛋白-紫杉醇纳米粒与药品Abraxane®的再分散Figure 6 shows the redispersion of albumin-paclitaxel nanoparticles and drug Abraxane®
其中，(a)2 mg/mL白蛋白-紫杉醇纳米粒溶液；(b)2 mg/mL Abraxane®溶液；(c)20 mg/mL白蛋白-紫杉醇纳米粒溶液；(d)20 mg/mL Abraxane®溶液；(e)50 mg/mL白蛋白-紫杉醇纳米粒溶液；(f)50 mg/mL Abraxane®溶液。Among them, (a) 2 mg/mL albumin-paclitaxel nanoparticle solution; (b) 2 mg/mL Abraxane® solution; (c) 20mg/mL albumin-paclitaxel nanoparticle solution; (d) 20mg/mL Abraxane® solution; (e) 50 mg/mL albumin-paclitaxel nanoparticle solution; (f) 50 mg/mLAbraxane® solution.
具体实施方式Detailed ways
以下均是基于本发明的代表性实施例，但下述实施例不会在任何方面限制本发明的保护范围。The following are all representative examples based on the present invention, but the following examples do not limit the protection scope of the present invention in any way.
实施例1.Example 1.紫杉醇paclitaxel--白蛋白纳米粒的制备Preparation of albumin nanoparticles
100 mgHSA溶于10 mL pH6的含0.5 mg/mL EDTA和0.05 M巯基乙醇的磷酸盐缓冲液，55℃反应持续两小时，结束后用5%的三氯乙酸沉淀并洗涤蛋白，加入1.6 mL10 mg/mL的紫杉醇（乙醇溶解）溶液到沉淀中，混合2分钟，加入50 mL的0.08 M的磷酸盐缓冲液搅拌，溶解混合物。得到的悬液透明，而且载药粒子的平均粒径为80～200 nm，(BIC 90plus Particle Size Analyzer)。用反相C18柱测定紫杉醇的包封率， 流动相为乙腈：水（60:40），检测波长227 nm。HPLC分析表明本实验的紫杉醇包封率达到90%以上。100 mgHSA was dissolved in 10 mL pH6 containing 0.5 mg/mL EDTA and 0.05M mercaptoethanol in phosphate buffer, react at 55°C for two hours, after the end, use 5% trichloroacetic acid to precipitate and wash the protein, add 1.6 mL of 10 mg/mL paclitaxel (dissolved in ethanol) solution to the precipitate, and mix for 2 minutes , add 50 mL of 0.08 M phosphate buffer and stir to dissolve the mixture. The obtained suspension is transparent, and the average particle size of the drug-loaded particles is 80-200nm, (BIC 90plus Particle Size Analyzer). The encapsulation efficiency of paclitaxel was determined by a reversed-phase C18 column, the mobile phase was acetonitrile: water (60:40), and the detection wavelength was 227 nm. HPLC analysis showed that the encapsulation efficiency of paclitaxel in this experiment was over 90%.
实验中三氯乙酸还可以用其他变性剂代替，比如水，强酸（盐酸和硫酸），强碱（氢氧化钠），过氧化氢，谷胱甘肽等。结果发现，5%的三氯乙酸可以达到较窄的粒径分布（50～300 nm）。In the experiment, TCA can also be replaced by other denaturants, such as water, strong acids (hydrochloric acid and sulfuric acid), strong bases (sodium hydroxide), hydrogen peroxide, glutathione, etc. It was found that 5% trichloroacetic acid can achieve a narrow particle size distribution (50-300nm).
实施例2.Example 2.紫杉醇Paclitaxel--白蛋白纳米粒的制备Preparation of albumin nanoparticles
100 mg HSA溶于50 mL pH7.4的TRIS缓冲液中，37℃水浴，加入350 µL 2-巯基乙醇，反应持续10分钟，加入2 mL 10 mg/mL的紫杉醇（乙醇溶解）。30分钟后，样品用pH7.4的TRIS缓冲液透析24小时，所得样品冻干48小时。所得冻干后的块状样品能够很容易的用水或生理盐水复溶为原溶液，且纳米粒粒径保持不变。冻干后粒径主要分布在80～200 nm(BIC 90plus Particle Size Analyzer)，HPLC分析表明本实验的紫杉醇包封率达到90%以上。100 mg HSA dissolved in 50 mLIn the TRIS buffer solution with pH 7.4, add 350 µL 2-mercaptoethanol in a 37°C water bath, react for 10 minutes, and add 2 mL of 10 mg/mL paclitaxel (dissolved in ethanol). After 30 min, samples were dialyzed against pH 7.4 TRIS buffer for 24 h and the resulting samples were lyophilized for 48 h. The resulting lyophilized block sample can be easily redissolved into the original solution with water or saline, and the particle size of the nanoparticles remains unchanged. After freeze-drying, the particle size is mainly distributed in the range of 80-200nm (BIC 90plus Particle Size Analyzer), HPLC analysis showed that the encapsulation efficiency of paclitaxel in this experiment reached more than 90%.
实施例3.Example 3.紫杉醇paclitaxel--白蛋白纳米粒的制备Preparation of albumin nanoparticles
100 mg紫杉醇溶解在10 mL pH4.8的缓冲液中，冰水浴30分钟，滴加7.5 mL 0℃预冷的丙酮，继续冰浴1小时。离心，收集沉淀，向沉淀中加入1 mL 10 mg/mL的紫杉醇（丙酮溶解），超声混匀，加入50 mL生理盐水，磁力搅拌，形成悬液。用BIC 90plus Particle Size Analyzer进行粒径分析，结果为150～220 nm。冻干的样品能复溶，HPLC分析载药量为8.34%。100 mg of paclitaxel was dissolved in 10 mL of pH 4.8 buffer solution, placed in an ice-water bath for 30 minutes, then 7.5 mL of 0°C pre-cooled acetone was added dropwise, and the ice bath was continued for 1 hour. Centrifuge, collect the precipitate, add 1 mL to the precipitate10 mg/mL paclitaxel (dissolved in acetone), ultrasonically mix, add 50 mL of normal saline, and magnetically stir to form a suspension. Particle size analysis was carried out with BIC 90plus Particle Size Analyzer, and the result was 150-220 nm. The freeze-dried sample could be reconstituted, and the drug loading was 8.34% according to HPLC analysis.
附加实验表明甘氨酸，甘露醇，乳糖和海藻糖均能作为冻干保护剂，且用乳糖做冻干保护剂得到的粒径最小。Additional experiments show that glycine, mannitol, lactose and trehalose can all be used as lyoprotectant, and the particle size obtained by using lactose as lyoprotectant is the smallest.
在制备过程中，我们考察了不同的缓冲液（水，生理盐水，磷酸盐缓冲液，醋酸盐缓冲液，甘氨酸，三羟甲基氨基甲烷，海藻糖，甘露醇，蔗糖，乙酰色氨酸，辛酸钠和葡萄糖溶液等）以及不同的pH对粒径和载药量的影响，结果表明pH在8.0较好。但是pH又不能超过8.5，因为药理活性物质在pH超过8.5的时候会分解。During the preparation, we investigated different buffers (water, saline, phosphate buffer, acetate buffer, glycine, tris, trehalose, mannitol, sucrose, acetyl tryptophan , sodium octanoate and glucose solution, etc.) and the effect of different pH on particle size and drug loading, the results showed that the pH is better at 8.0. But the pH cannot exceed 8.5, because the pharmacologically active substance will decompose when the pH exceeds 8.5.
实施例4.Example 4.紫杉醇paclitaxel--转铁蛋白纳米粒的制备Preparation of transferrin nanoparticles
100 mg转铁蛋白溶于50 mL pH7.4的TRIS缓冲液中，75℃搅拌，加入350 µL 2-巯基乙醇，反应持续10分钟，缓慢加入1 mL 10 mg/mL的紫杉醇（乙醇溶解）。测得粒径分布在154.4 nm（BIC 90plus Particle Size Analyzer）。100 mg of transferrin was dissolved in 50 mL of TRIS buffer at pH 7.4, stirred at 75°C, 350 µL of 2-mercaptoethanol was added, the reaction was continued for 10 minutes, and 1 mL of 10 mg/mL paclitaxel (dissolved in ethanol) was slowly added. The measured particle size distribution is 154.4nm (BIC 90plus Particle Size Analyzer).
在制备过程中，我们考察了不同温度对粒径和载药量的影响，结果表明温度在0℃到100℃之间均能够形成纳米粒，在55～75℃形成的纳米粒更好。During the preparation process, we investigated the effect of different temperatures on particle size and drug loading, and the results showed that nanoparticles could be formed at temperatures between 0°C and 100°C, and nanoparticles formed at 55-75°C were better.
实施例5.Example 5.多烯紫杉醇Docetaxel--转铁蛋白纳米粒的制备Preparation of transferrin nanoparticles
100 mg转铁蛋白溶于50 mL pH7.4的TRIS缓冲液中，65℃搅拌，加入350 µL 2-巯基乙醇，反应持续10分钟，加入5 mL 10 mg/mL的多烯紫杉醇（乙醇溶解）。测得平均粒径为177.1 nm（BIC 90plus Particle Size Analyzer）。Dissolve 100 mg of transferrin in 50 mL of TRIS buffer at pH 7.4, stir at 65°C, add 350 µL of 2-mercaptoethanol, react for 10 minutes, add 5 mL of 10 mg/mL docetaxel (dissolved in ethanol) . The measured average particle size is 177.1nm (BIC 90plus Particle Size Analyzer).
若2-巯基乙醇和其他无机盐配合使用，可以得到更好的粒径结果，比如水，氯化钠，磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷，过氧化氢，谷胱甘肽，葡萄糖，蔗糖，甘露醇，海藻糖，乙酰色氨酸和辛酸钠等。 当这些无机盐与2-巯基乙醇混合作为变性剂时，粒子直径分布更窄，其中，三羟甲基氨基甲烷，乙酰色氨酸和辛酸钠的粒子分布最窄，大约为80～130 nm。If 2-mercaptoethanol is used in combination with other inorganic salts, better particle size results can be obtained, such as water, sodium chloride, phosphate, acetate, glycine, tris, hydrogen peroxide, glutathione Glycosin, glucose, sucrose, mannitol, trehalose, acetyl tryptophan and sodium caprylate etc. When these inorganic salts are mixed with 2-mercaptoethanol as a denaturant, the particle diameter distribution is narrower, among which, the particle distribution of trishydroxymethylaminomethane, acetyl tryptophan and sodium octanoate is the narrowest, about 80-130nm.
实施例6.Example 6.藤黄酸Gambogic acid--白蛋白纳米粒的制备Preparation of albumin nanoparticles
100 mg HSA溶于10 mL纯水中。55℃搅拌，所得溶液和等体积的5%三氯乙酸溶液混合并且离心，除去上清。加入含有藤黄酸的乙醇溶液混合，然后加入50 mL pH8.0的TRIS，搅拌直至蛋白沉淀完全溶解。所得的纳米粒粒径为110 nm（BIC 90plus Particle Size Analyzer）。100 mg HSA was dissolved in 10 mL pure water. Stirring at 55°C, the resulting solution was mixed with an equal volume of 5% trichloroacetic acid solution and centrifuged to remove the supernatant. Add an ethanol solution containing gambogic acid and mix, then add 50 mL of TRIS at pH 8.0, and stir until the protein precipitate is completely dissolved. The obtained nanoparticles have a particle size of 110nm (BIC 90plus Particle Size Analyzer).
实施例7.Example 7.紫杉醇paclitaxel--血红蛋白纳米粒的制备Preparation of hemoglobin nanoparticles
-20℃条件下，向10 mL3%的血红蛋白水溶液中缓慢加入300 mL含有3 mL 2 M的HCl的丙酮溶液。溶液强力搅拌15分钟，然后离心15分钟。待残余的丙酮蒸发掉以后，收集沉淀，溶解在冷的去离子水里，在2℃条件下用去离子水透析5个小时，然后用0.0016 M NaHCO3透析30小时，过滤得到珠蛋白溶液。At -20°C, slowly add 300 mL of acetone solution containing 3 mL of 2 M HCl to 10 mL of 3% hemoglobin aqueous solution. The solution was stirred vigorously for 15 minutes and then centrifuged for 15 minutes. After the residual acetone evaporated, the precipitate was collected, dissolved in cold deionized water, dialyzed against deionized water at 2°C for 5 hours, then dialyzed against 0.0016 M NaHCO for30 hours, and filtered to obtain a globin solution.
取7 mL上述的珠蛋白溶液加入28 mL水，得到1 mg/mL的蛋白溶液。在2～8℃条件下加入0.7 mL含10 mg/mL紫杉醇的乙醇溶液，搅拌直至形成浅蓝色溶液。所得粒子平均粒径为308.1 nm（BIC 90plus Particle Size Analyzer）。Take 7 mL of the above globin solution and add 28 mL of water to obtain a 1 mg/mL protein solution. Add 0.7 mL of ethanol solution containing 10 mg/mL paclitaxel at 2-8 °C, and stir until a light blue solution is formed. The average particle diameter of the obtained particles was 308.1 nm (BIC 90plus Particle Size Analyzer).
实施例8.Example 8.pHpH对纳米粒粒径的影响考察Influence on the particle size of nanoparticles
本发明考察了不同pH的磷酸盐缓冲液以及它们对纳米粒粒径的影响。所有的蛋白溶液组中均加入15%的紫杉醇(10 mg/mL)，粒径在恒温条件下测定，结果如图1所示。The present invention investigates phosphate buffers with different pHs and their effects on the particle size of nanoparticles. 15% paclitaxel (10 mg/mL) was added to all protein solution groups, and the particle size was measured under constant temperature conditions. The results are shown in Figure 1.
实施例9.Example 9.紫杉醇paclitaxel--白蛋白纳米粒的动态光散射Dynamic Light Scattering of Albumin Nanoparticles((dynamic light scaterringdynamic light scattering,,DLSDLS))分析analyze
对按照本发明的方法制备的紫杉醇-白蛋白纳米粒进行粒径分析，所用仪器为BIC 90plus Particle Size Analyzer。结果如图4所示，平均粒径为121 nm，且粒径分布在较窄的范围内。Carry out particle size analysis to paclitaxel-albumin nanoparticles prepared according to the method of the present invention, the instrument used is BIC90plus Particle Size Analyzer. The results are shown in Figure 4, the average particle size is 121 nm, and the particle size distribution is in a narrow range.
实施例10.Example 10.紫杉醇paclitaxel--白蛋白纳米粒的透射电镜TEM of albumin nanoparticles(Transmission Electron Microscopy, TEM)(Transmission Electron Microscopy, TEM)表征characterize
按照本发明制备载药量为10.59%的紫杉醇-白蛋白纳米粒进行透射电镜的分析。所用仪器为EM 2100型200KV高分辨率透射电镜(日本)。结果如图2所示，显示纳米粒子呈球形。Paclitaxel-albumin nanoparticles with a drug loading of 10.59% were prepared according to the present invention and analyzed by a transmission electron microscope. The instrument used is EM 2100 200KV high-resolution transmission electron microscope (Japan). The results are shown in Figure 2, showing that the nanoparticles were spherical.
实施例11.Example 11.复溶的紫杉醇reconstituted paclitaxel--白蛋白纳米粒的稳定性研究Study on Stability of Albumin Nanoparticles
冻干得到的紫杉醇-白蛋白块状物用生理盐水，5%的葡萄糖和小牛血清分别溶解成紫杉醇浓度为5 mg/mL，5 mg/mL和2 mg/mL的溶液。分别在25℃和37℃放置12小时后，纳米粒的平均粒径没有变化（DLS, BIC 90plus Particle Size Analyzer），且没有沉淀产生。结果如表1所示。The paclitaxel-albumin block obtained by freeze-drying was dissolved in normal saline, 5% glucose and calf serum respectively so that the concentration of paclitaxel was 5 mg/mL, 5mg/mL and 2 mg/mL solutions. After being placed at 25°C and 37°C for 12 hours, the average particle size of the nanoparticles did not change (DLS, BIC 90plus Particle Size Analyzer), and no precipitation occurred. The results are shown in Table 1.
表 1 紫杉醇-白蛋白纳米粒的稳定性研究Table 1 Stability studies of paclitaxel-albumin nanoparticles
实施例12.Example 12.紫杉醇paclitaxel--转铁蛋白纳米粒的研究Study on Transferrin Nanoparticles
在制备紫杉醇-转铁蛋白纳米粒的过程中，发现除了TRIS缓冲液，生理盐水也可以用来溶解转铁蛋白。另外，在pH为3～9，特别是在6～8范围内可以获得高载药量和高稳定性的纳米粒。而且，蔗糖，葡萄糖，甘氨酸和海藻糖可以用作冻干或减压干燥保护剂。In the process of preparing paclitaxel-transferrin nanoparticles, it was found that in addition to TRIS buffer, normal saline can also be used to dissolve transferrin. In addition, nanoparticles with high drug loading and high stability can be obtained at a pH of 3-9, especially in the range of 6-8. Also, sucrose, glucose, glycine, and trehalose can be used as protectants for freeze-drying or reduced-pressure drying.
实施例13.Example 13.复溶的紫杉醇reconstituted paclitaxel--转铁蛋白纳米粒的稳定性研究Study on Stability of Transferrin Nanoparticles
冻干得到的紫杉醇--转铁蛋白块状物用生理盐水，5%的葡萄糖和小牛血清分别溶解成紫杉醇浓度为5 mg/mL，5 mg/mL和2 mg/mL的溶液。分别在25℃和37℃放置12小时后，纳米粒的平均粒径没有变化（DLS, BIC 90plus Particle Size Analyzer），且没有沉淀产生。结果如表2所示。The paclitaxel obtained by lyophilization--transferrin block was dissolved in normal saline, 5% glucose and calf serum respectively so that the concentration of paclitaxel was 5 mg/mL, 5mg/mL and 2 mg/mL solutions. After being placed at 25°C and 37°C for 12 hours, the average particle size of the nanoparticles did not change (DLS, BIC 90plus Particle Size Analyzer), and no precipitation occurred. The results are shown in Table 2.
表 2紫杉醇-转铁蛋白纳米粒的稳定性研究Table 2 Stability studies of paclitaxel-transferrin nanoparticles
实施例14.Example 14.用透射电镜TEM(Transmission Electron Microscopy, TEM)(Transmission Electron Microscopy, TEM)对紫杉醇paclitaxel--白蛋白纳米粒进行的表征Characterization of Albumin Nanoparticles
将2～3滴按照本发明制备的紫杉醇-白蛋白纳米粒溶液滴在覆盖有碳支持膜的铜网上(200目)，2分钟后用滤纸吸干多余的溶液，然后将铜网放在空气中干燥，用EM-2100 200 KV型高分辨率透射电镜（日本）进行分析。Drop 2 to 3 drops of the paclitaxel-albumin nanoparticle solution prepared according to the present invention on a copper grid (200 mesh) covered with a carbon support film, blot the excess solution with filter paper after 2 minutes, and then place the copper grid in the air Medium dry, use EM-2100 200KV-type high-resolution transmission electron microscope (Japan) for analysis.
结果如图4所示：a)紫杉醇-白蛋白纳米粒呈球形；b)空白的白蛋白纳米粒呈现不规则的形状，且平均粒径在100 nm左右；c)游离的紫杉醇在中间呈现处较高的电子密度，周围被棒状结构包围；d)紫杉醇-白蛋白纳米粒呈现出核壳结构。The results are shown in Figure 4: a) paclitaxel-albumin nanoparticles are spherical; b) blank albumin nanoparticles are irregular in shape, and the average particle size is about 100 nm; c) free paclitaxel appears in the middle Higher electron density, surrounded by rod-like structures; d) Paclitaxel-albumin nanoparticles present a core-shell structure.
实施例15.Example 15.紫杉醇Paclitaxel--白蛋白纳米粒的albumin nanoparticlesXx射线粉末衍射ray powder diffraction(x-ray powder diffraction, XRD)(x-raypowder diffraction, XRD)表征characterize
紫杉醇在水溶液中超过1 mg/mL时就会结晶。因此，无定形态的紫杉醇非常有利于注射。为了检测本发明中紫杉醇-白蛋白纳米粒中紫杉醇的固体形态，我们使用了X射线粉末衍射来进行表征。共制备了四个样品：a)紫杉醇；b)白蛋白纳米粒；c)紫杉醇白蛋白纳米粒（载药量为12.9%）；d)紫杉醇和白蛋白的物理混合物（12.9%）。每个样品的Zeta角测定范围从5度到50度（ARL, X'TRA ,Applied Research Laboratories, Switzerland）。Paclitaxel crystallizes above 1 mg/mL in aqueous solution. Therefore, the amorphous form of paclitaxel is very convenient for injection. In order to detect the solid form of paclitaxel in the paclitaxel-albumin nanoparticles of the present invention, we used X-ray powder diffraction for characterization. A total of four samples were prepared: a) paclitaxel; b) albumin nanoparticles; c) paclitaxel-albumin nanoparticles (12.9% drug loading); d) physical mixture of paclitaxel and albumin (12.9%). Zeta angles were measured for each sample ranging from 5° to 50° (ARL, X'TRA , Applied Research Laboratories, Switzerland).
结果如图5所示：图a)显示了紫杉醇晶体的特征峰；在2θ角为15度到45度范围内，图b)显示出了白蛋白的晕型峰；图c)表明在2θ角为15度到45度范围内，也显示了白蛋白的晕型峰，说明在纳米粒中紫杉醇是以无定形的状态存在；在与纳米粒相同比例（12.9%）的紫杉醇和白蛋白的物理混合物中，图d)显示了晶体状态的紫杉醇和无定形状态的白蛋白的存在。因此，我们可以得到结论：本发明制备的紫杉醇-白蛋白纳米中紫杉醇以无定形状态存在。The results are shown in Figure 5: Figure a) shows the characteristic peaks of paclitaxel crystals; in the range of 15 degrees to 45 degrees at the 2θ angle, Figure b) shows the halo peak of albumin; Figure c) shows that at the 2θ angle In the range of 15 degrees to 45 degrees, it also shows the halo peak of albumin, indicating that paclitaxel exists in an amorphous state in nanoparticles; In the mixture, panel d) shows the presence of paclitaxel in the crystalline state and albumin in the amorphous state. Therefore, we can conclude that paclitaxel exists in an amorphous state in the paclitaxel-albumin nanoparticles prepared in the present invention.
实施例16.Example 16.紫杉醇Paclitaxel--白蛋白纳米粒的再分散研究Study on Redispersion of Albumin Nanoparticles
按照本发明制备的紫杉醇-白蛋白纳米粒冻干后的样品和商业化的药理活性物质Abraxane®按如下蛋白浓度用水溶解：(a, b) 2 mg/mL; (c,d) 20 mg/mL; (e,f) 50 mg/mL。这些样品的照片如图6所示，三个样品中均可以得到稳定的透明的紫杉醇-白蛋白纳米粒的胶体溶液。The paclitaxel-albumin nanoparticles lyophilized samples prepared according to the present invention and the commercialized pharmacologically active substance Abraxane® were dissolved in water according to the following protein concentration: (a, b) 2 mg/mL; (c, d) 20 mg/mL mL; (e,f) 50 mg/mL. The photos of these samples are shown in FIG. 6 , and stable and transparent colloidal solutions of paclitaxel-albumin nanoparticles can be obtained in the three samples.
实施例17.Example 17.伊立替康irinotecan--胰岛素纳米粒的制备Preparation of insulin nanoparticles
100 mg胰岛素溶于10 mL 水或者生理盐水中，65℃搅拌，结束后用甲醇沉淀并洗涤蛋白，加入1.6 mL10 mg/mL的伊立替康溶液到沉淀中，混合2分钟，加入50 mL的氯化钠溶液搅拌，溶解混合物。得到的载药粒子的直径最大为2000 nm，最小为5 nm，主要分布的范围在50～300 nm，部分纳米粒分布在2000 nm (BIC 90plus Particle Size Analyzer)。用反相C18柱测定的包封率， 流动相为乙腈：水（60:40），检测波长227 nm。HPLC分析表明本实验的伊立替康载药为2%。Dissolve 100 mg of insulin in 10 mL of water or normal saline, stir at 65°C, precipitate and wash the protein with methanol, add 1.6Add 10 mg/mL of irinotecan solution to the precipitate, mix for 2 minutes, add 50 mL of sodium chloride solution and stir to dissolve the mixture. The maximum diameter of the obtained drug-loaded particles is 2000 nm, the minimum is 5 nm, the main distribution range is 50-300 nm, and some nanoparticles are distributed at 2000 nm (BIC 90plus Particle Size Analyzer). The encapsulation efficiency was determined by a reversed-phase C18 column, the mobile phase was acetonitrile: water (60:40), and the detection wavelength was 227 nm. HPLC analysis showed that the drug loading of irinotecan in this experiment was 2%.
实验发现除了水或者生理盐水溶解胰岛素之外，一些蛋白稳定剂和冻干保护剂也可以用来溶解胰岛素。比如甘氨酸，谷胱甘肽，乙酰色氨酸，辛酸钠以及甘露醇等。另外，在反应温度为0℃～100℃，特别是在55～75℃范围内可以获得高载药量和高稳定性的纳米粒。得到的蛋白纳米粒可以用喷雾干燥的方式得到干性粉末。Experiments have found that in addition to dissolving insulin in water or saline, some protein stabilizers and lyoprotectants can also be used to dissolve insulin. Such as glycine, glutathione, acetyl tryptophan, sodium caprylate and mannitol. In addition, nanoparticles with high drug loading and high stability can be obtained at a reaction temperature of 0°C to 100°C, especially in the range of 55°C to 75°C. The obtained protein nanoparticles can be spray-dried to obtain dry powder.
实施例18.Example 18.5-5-氟尿嘧啶Fluorouracil--血管内皮抑素纳米粒的制备Preparation of Endostatin Nanoparticles
100 mg血管内皮抑素溶于10 mL pH 9的的含有的5%葡萄糖的溶液中，75℃水浴，加入25 mL 0.5 mg/mL的谷胱甘肽，反应10分钟，加入1.6 mL10 mg/mL的5-氟尿嘧啶，反应持续两小时。样品用pH7.4的TRIS缓冲液透析24小时，所得样品进行喷雾干燥后纳米粒粒径保持不变。粒径主要分布在25～500 nm(BIC 90plus Particle Size Analyzer)，载药量达到40%。Dissolve 100 mg of vascular endostatin in 10 mL of pH 9 solution containing 5% glucose, in a water bath at 75°C, add 25 mL of 0.5 mg/mL glutathione, react for 10 minutes, add 1.6 mL of 10 mg/mL 5-fluorouracil, the reaction lasted two hours. The sample was dialyzed with pH7.4 TRIS buffer for 24 hours, and the particle size of the nanoparticles remained unchanged after the obtained sample was spray-dried. The particle size is mainly distributed in the range of 25 to 500nm (BIC 90plus Particle Size Analyzer), the drug loading reached 40%.
其中，5%的葡萄糖可以换成是冻干保护剂中的任何一种，如磷酸盐，醋酸盐，甘氨酸，三羟甲基氨基甲烷。谷胱甘肽可以是结构展开剂中的任何一种，如2-巯基乙醇，二硫苏糖醇，盐酸胍，尿素，高氯酸，三丁基膦，甲巯丙脯氨酸，过甲酸，青霉胺，谷胱甘肽，甲硫咪唑，乙酰半胱氨酸或它们的组合。结果显示，溶解蛋白质的溶液对最后粒子的大小没有影响。变性剂或者适合的变性条件中，2-巯基乙醇的效果最好，得到的纳米粒子分布较窄，50～300 nm之间。Among them, 5% of the glucose can be replaced by any one of the lyoprotectants, such as phosphate, acetate, glycine, and tris. Glutathione can be any of the structure-expanding agents, such as 2-mercaptoethanol, dithiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, captopril, performic acid , penicillamine, glutathione, methimazole, acetylcysteine, or combinations thereof. The results showed that the solution in which the protein was dissolved had no effect on the final particle size. Among the denaturants or suitable denaturing conditions, 2-mercaptoethanol has the best effect, and the distribution of the obtained nanoparticles is narrow, between 50 and 300 nm.
实施例19.Example 19.5-5-氟尿嘧啶Fluorouracil--紫杉醇Paclitaxel--血管内皮抑素纳米粒的制备Preparation of Endostatin Nanoparticles
100 mg血管内皮抑素溶于10 mL pH7.4的的含有的5%海藻糖的溶液中，25℃水浴，加入25 mL 0.5 mg/mL的巯基乙醇和6 M的尿素，反应10分钟，加入0.8 mL10 mg/mL的5-氟尿嘧啶和0.8 mL10 mg/mL紫杉醇，持续搅拌，溶液呈淡蓝色。粒径主要分布在25～500 nm(BIC 90plus Particle Size Analyzer)。Dissolve 100 mg of vascular endostatin in 10 mL of pH7.4 solution containing 5% trehalose, in a water bath at 25°C, add 25 mL of 0.5 mg/mL mercaptoethanol and 6 M urea, react for 10 minutes, add 0.8 mL of 10 mg/mL 5-fluorouracil and 0.8 mL of 10 mg/mL paclitaxel were stirred continuously, and the solution was light blue. The particle size is mainly distributed in the range of 25 to 500nm (BIC 90plus Particle Size Analyzer).
其中，5%的葡萄糖可以换成是蛋白稳定剂中的任何一种，如甘露醇，蔗糖，乙酰色氨酸，辛酸钠。结果显示，溶解蛋白质的溶液对最后粒子的大小没有影响，得到的纳米粒子分布较窄，50～300 nm之间。Among them, 5% of glucose can be replaced by any one of protein stabilizers, such as mannitol, sucrose, acetyl tryptophan, sodium caprylate. The results showed that the solution in which the protein was dissolved had no effect on the size of the final particles, and the distribution of the obtained nanoparticles was narrow, 50-300between nm.
实施例20.Example 20.卡莫司汀Carmustine--肌红蛋白纳米粒的制备Preparation of myoglobin nanoparticles
100 mg肌红蛋白溶于10 mL pH 3的含0.5 mg/mL 辛酸钠和0.05 M乙酰色氨酸的磷酸盐缓冲液，冰浴30分钟，滴加0℃预冷的丙酮7.5 mL。继续冰浴1小时，离心，收集沉淀，向沉淀中加入1.6 mL 10 mg/mL的卡莫司汀溶液，超声混匀，加入50 mL Tris缓冲液，磁力搅拌，得到的悬液透明，溶液经过减压蒸馏得到平均粒径为80～200 nm的粒子(BIC 90plus Particle Size Analyzer),100 mg myoglobin dissolved in 10 mL pH 3 containing 0.5 mg/mL sodium caprylate and 0.05M Acetyl tryptophan in phosphate buffer, ice-bathed for 30 minutes, and 7.5 mL of acetone pre-cooled at 0°C was added dropwise. Continue the ice bath for 1 hour, centrifuge, collect the precipitate, add 1.6 mL of 10 mg/mL carmustine solution to the precipitate, ultrasonically mix, add 50 mL of Tris buffer, and magnetically stir, the obtained suspension is transparent, and the solution passes through Particles (BIC 90plusParticle Size Analyzer),
其中变性条件丙酮可以替换成为其他能够使蛋白变性的有机溶剂，如甲醇，乙醇，异丙醇，福尔马林，氯仿，硫化氢或者他们的组合。结果显示，变性条件若使用有机溶剂，则丙酮的效果最好，粒子直径分布在50～300 nm之间。Among the denaturing conditions, acetone can be replaced by other organic solvents that can denature proteins, such as methanol, ethanol, isopropanol, formalin, chloroform, hydrogen sulfide or their combination. The results show that if the denaturation condition uses an organic solvent, the effect of acetone is the best, and the particle diameter distribution is between 50 and 300between nm.
实施例21.Example 21.阿霉素Adriamycin--溶菌酶纳米粒的制备Preparation of Lysozyme Nanoparticles
100 mg溶菌酶溶于50 mL pH 6的TRIS缓冲液中，55℃搅拌，通入硫化氢气体，反应持续10分钟，加入5 mL 10 mg/mL的阿霉素。测得粒径分布在347.1 nm（BIC 90plus Particle Size Analyzer）。Dissolve 100 mg of lysozyme in 50 mL of TRIS buffer at pH 6, stir at 55 °C, pass through hydrogen sulfide gas, react for 10 minutes, and add 5 mL of 10 mg/mL doxorubicin. The particle size distribution was measured at 347.1 nm (BIC90plus Particle Size Analyzer).
Tris缓冲液可以换成蛋白稳定剂中的任何一种，如海藻糖，甘露醇，蔗糖，乙酰色氨酸，辛酸钠或它们的组合。Tris buffer can be replaced with any of the protein stabilizers such as trehalose, mannitol, sucrose, acetyl tryptophan, sodium caprylate or their combination.
实施例22.Example 22.苯芥胆甾醇Cholesterol--免疫球蛋白纳米粒的制备Preparation of immunoglobulin nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为苯芥胆甾醇，转铁蛋白为免疫球蛋白。平均粒径大约为250 nm，载药量9.8%。This example is the same as Example 5, except that the docetaxel is changed to bensterol, and the transferrin is changed to immunoglobulin. The average particle size is about 250nm, drug loading 9.8%.
实施例23.Example 23.哌泊舒凡Pibosufan--αalpha-2--2-巨球蛋白纳米粒的制备Preparation of macroglobulin nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为哌泊舒凡，转铁蛋白为α-2-巨球蛋白，反应温度为65℃。粒径分布25 nm～500 nm， 载药量11.2%。This example is the same as Example 5, except that docetaxel is changed to pibosulfan, transferrin is changed to α-2-macroglobulin, and the reaction temperature is 65°C. Particle size distribution 25 nm～500nm, drug loading 11.2%.
实施例24.Example 24.他莫昔芬Tamoxifen--纤维连接蛋白纳米粒的制备Preparation of Fibronectin Nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为他莫昔芬，转铁蛋白为纤维连接蛋白。反应温度45℃，粒径分布50 nm～200 nm，载药量9.8%。This example is the same as Example 5, except that docetaxel is changed to tamoxifen, and transferrin is changed to fibronectin. The reaction temperature is 45°C, the particle size distribution is 50 nm to 200 nm, and the drug loading is 9.8%.
实施例25.Example 25.洛莫司汀Lomustine--纤层蛋白纳米粒的制备Preparation of lamin nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为洛莫司汀，转铁蛋白为纤层蛋白。反应温度为75℃，粒径分布在100 nm～200 nm之间，载药量35%。This example is the same as Example 5, except that docetaxel is changed to lomustine, and transferrin is changed to lamin. The reaction temperature is 75°C, the particle size distribution is between 100 nm and 200 nm, and the drug loading is 35%.
实施例26.Example 26.冬凌草甲素Oridonin A--胶原蛋白纳米粒的制备Preparation of Collagen Nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为冬凌草甲素，转铁蛋白为胶原蛋白。反应的pH为8.0，粒径分布在300 nm～600 nm之间，载药量22%。This example is the same as Example 5, except that docetaxel is changed to oridonin, and transferrin is changed to collagen. The pH of the reaction is 8.0, and the particle size distribution is between 300 nm and 600nm, the drug loading is 22%.
实施例27.Example 27.鬼臼毒素podophyllotoxin--明胶纳米粒的制备Preparation of gelatin nanoparticles
本实施例与实施例5相同，仅改变多西紫杉醇为鬼臼毒素，转铁蛋白为明胶。反应的温度为70℃，粒径分布在50 nm～300 nm之间，载药量20%。This example is the same as Example 5, except that docetaxel is changed to podophyllotoxin, and transferrin is changed to gelatin. The reaction temperature is 70°C, the particle size distribution is between 50 nm and 300 nm, and the drug loading is 20%.
实施例28.Example 28.阿托伐他汀Atorvastatin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
100 mgHSA溶于10 mL生理盐水中，含0.5 mg/mL 辛酸钠和0.05 M乙酰色氨酸，55℃水浴。加入350 µL 2-巯基乙醇，反应持续10 min，加入2 mL 10 mg/mL的阿托伐他汀，继续搅拌直到溶液呈淡蓝色，所得的纳米粒粒径为50～300 nm（BIC 90plus Particle Size Analyzer），载药量为24%。100 mgHSA dissolved in 10 mL of normal saline containing 0.5 mg/mL sodium caprylate and 0.05M Acetyl tryptophan, 55°C water bath. join 350µL of 2-mercaptoethanol, the reaction lasted 10 min, and 2 mL10 mg/mL atorvastatin, continue to stir until the solution is light blue, and the obtained nanoparticles have a particle size of 50-300nm (BIC 90plus Particle Size Analyzer), the drug loading is 24%.
实施例29.Example 29.斯伐他汀Simvastatin--转铁蛋白纳米粒的制备Preparation of transferrin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为斯伐他汀，白蛋白为转铁蛋白。所得纳米颗粒粒径为50～300 nm，载药量为5%。This example is the same as Example 28, except that atorvastatin is changed to simvastatin, and albumin is changed to transferrin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 5%.
实施例30.Example 30.菲诺贝特Finobate--血红蛋白纳米粒的制备Preparation of hemoglobin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为菲诺贝特，白蛋白为血红蛋白。所得纳米颗粒粒径为30～300 nm，载药量为11%。This example is the same as Example 28, except that atorvastatin is changed to fenofibrate, and albumin is changed to hemoglobin. The particle size of the obtained nanoparticles is 30-300nm, the drug loading was 11%.
实施例31.Example 31.硝苯地平Nifedipine--血管内皮抑素纳米粒的制备Preparation of Endostatin Nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为硝苯地平，白蛋白为血管内皮抑素。所得纳米颗粒粒径为20～250 nm，载药量为9.7%。This example is the same as Example 28, except that atorvastatin is changed to nifedipine, and albumin is changed to endostatin. The particle size of the obtained nanoparticles is 20-250nm, the drug loading was 9.7%.
实施例32.Example 32.布洛芬ibuprofen--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为布洛芬。所得纳米颗粒粒径为20～250 nm，载药量为8.7%。This example is the same as Example 28, except that atorvastatin is changed to ibuprofen. The particle size of the obtained nanoparticles is 20-250nm, the drug loading was 8.7%.
实施例33.Example 33.吲哚美辛Indomethacin--胶原蛋白纳米粒的制备Preparation of Collagen Nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为吲哚美辛，白蛋白为胶原蛋白。所得纳米颗粒的粒径为30～500 nm，载药量为8.9%。This example is the same as Example 28, except that atorvastatin is changed to indomethacin, and albumin is changed to collagen. The particle size of the obtained nanoparticles is 30-500nm, the drug loading was 8.9%.
实施例34.Example 34.吡罗昔康piroxicam--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为吡罗昔康。所得纳米颗粒的粒径为25～500 nm，载药量为8.0%。This example is the same as Example 28, except that atorvastatin is changed to piroxicam. The particle size of the obtained nanoparticles is 25～500nm, the drug loading is 8.0%.
实施例35.Example 35.格列苯脲Glibenclamide--肌红蛋白纳米粒的制备Preparation of myoglobin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为格列苯脲，白蛋白为肌红蛋白。所得纳米颗粒的粒径为50～300 nm，载药量为7.8%。This example is the same as Example 28, except that atorvastatin is changed to glibenclamide, and albumin is changed to myoglobin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 7.8%.
实施例36.Example 36.地西泮Diazepam--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为地西泮。所得纳米颗粒的粒径为50～300 nm，载药量为8.8%。This example is the same as Example 28, except that atorvastatin is changed to diazepam. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 8.8%.
实施例37.Example 37.利哌利酮Risperidone--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为利哌利酮。所得纳米颗粒的粒径为50～300 nm，载药量为9.8%。This example is the same as Example 28, only atorvastatin is changed to risperidone. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 9.8%.
实施例38.Example 38.齐拉西酮Ziprasidone--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为齐拉西酮, 白蛋白为免疫球蛋白，所得纳米颗粒的粒径为50～300 nm，载药量为7.3%。This example is the same as Example 28, except that atorvastatin is changed to ziprasidone, albumin is changed to immunoglobulin, the particle size of the obtained nanoparticles is 50-300 nm, and the drug loading is 7.3%.
实施例39.Example 39.他克莫司Tacrolimus--溶菌酶纳米粒的制备Preparation of Lysozyme Nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为他克莫司，白蛋白为溶菌酶。所得纳米颗粒的粒径为50～300 nm，载药量为12.8%。This example is the same as Example 28, except that atorvastatin is changed to tacrolimus, and albumin is changed to lysozyme. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 12.8%.
实施例40.Example 40.雷帕霉素Rapamycin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为雷帕霉素。所得纳米颗粒的粒径为50～300 nm，载药量为9.8%。This example is the same as Example 28, only atorvastatin is changed to rapamycin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 9.8%.
实施例41.Example 41.茚地那韦Indinavir--转铁蛋白纳米粒的制备Preparation of transferrin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为茚地那韦，白蛋白为转铁蛋白。所得纳米颗粒的粒径为50～300 nm，载药量为13.5%。This example is the same as Example 28, except that atorvastatin is changed to indinavir, and albumin is changed to transferrin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 13.5%.
实施例42.Example 42.利托那韦Ritonavir--胰岛素纳米粒的制备Preparation of insulin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为利托那韦，白蛋白为胰岛素。所得纳米颗粒的粒径为50～300 nm，载药量为9.8%。This example is the same as Example 28, except that atorvastatin is changed to ritonavir, and albumin is changed to insulin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 9.8%.
实施例43.Example 43.洛匹那韦Lopinavir--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为洛匹那韦。所得纳米颗粒的粒径为50～300 nm，载药量为11.3%。This example is the same as Example 28, only changing atorvastatin to lopinavir. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 11.3%.
实施例44.Example 44.环磷酰胺cyclophosphamide--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为环磷酰胺。所得纳米颗粒的粒径为50～300 nm，载药量为11.9%。This example is the same as Example 28, only changing atorvastatin to cyclophosphamide. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 11.9%.
实施例45.Example 45.博来霉素Bleomycin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为博来霉素。所得纳米颗粒的粒径为50～300 nm，载药量为12.8%。This example is the same as Example 28, only atorvastatin is changed to bleomycin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 12.8%.
实施例46.Example 46.道诺霉素daunomycin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为道诺霉素。所得纳米颗粒的粒径为50～300 nm，载药量为13.8%。This example is the same as Example 28, only changing atorvastatin to daunomycin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 13.8%.
实施例47.Example 47.表柔比星Epirubicin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为表柔比星。所得纳米颗粒的粒径为50～300 nm，载药量为7.3%。This example is the same as Example 28, except that atorvastatin is changed to epirubicin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 7.3%.
实施例48.Example 48.甲氨蝶呤methotrexate--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为甲胺蝶呤。所得纳米颗粒的粒径为50～300 nm，载药量为6.8%。This example is the same as Example 28, only atorvastatin is changed to methotrexate. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 6.8%.
实施例49.Example 49.5-5-氟尿嘧啶Fluorouracil--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为氟尿嘧啶。所得纳米颗粒的粒径为50～300 nm，载药量为11.1%。This example is the same as Example 28, only changing atorvastatin to fluorouracil. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 11.1%.
实施例50.Example 50.顺铂Cisplatin--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为顺铂。所得纳米颗粒的粒径为50～300 nm，载药量为12.8%。This example is the same as Example 28, only changing atorvastatin to cisplatin. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 12.8%.
实施例51.Example 51.长春碱Vinblastine--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为长春碱。所得纳米颗粒的粒径为50～300 nm，载药量为6.3%。This example is the same as Example 28, only atorvastatin is changed to vinblastine. The particle size of the obtained nanoparticles is 50-300nm, the drug loading is 6.3%.
实施例52.Example 52.放线菌素ActinomycinD-D-白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为放线菌素D。所得纳米颗粒的粒径为50～300 nm，载药量为5.8%。This example is the same as Example 28, only atorvastatin is changed to actinomycin D. The particle size of the obtained nanoparticles is 50-300 nm, and the drug loading capacity is 5.8%.
实施例53.Example 53.丝裂霉素mitomycinC-C-白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为丝裂霉素C。所得纳米颗粒的粒径为50～300 nm，载药量为4.8%。This example is the same as Example 28, only changing atorvastatin to mitomycin C. The particle size of the obtained nanoparticles is 50-300 nm, and the drug loading capacity is 4.8%.
实施例54.Example 54.依托泊苷Etoposide--白蛋白纳米粒的制备Preparation of albumin nanoparticles
本实施例与实施例28相同，仅改变阿托伐他汀为依托泊苷。所得纳米颗粒的粒径为50～300 nm，载药量为14.9%。This example is the same as Example 28, only changing atorvastatin to etoposide. The particle size of the obtained nanoparticles is 50-300nm, the drug loading was 14.9%.
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