CN101745115B - 酪蛋白纳米粒子 - Google Patents
酪蛋白纳米粒子 Download PDFInfo
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- CN101745115B CN101745115B CN2009102531362A CN200910253136A CN101745115B CN 101745115 B CN101745115 B CN 101745115B CN 2009102531362 A CN2009102531362 A CN 2009102531362A CN 200910253136 A CN200910253136 A CN 200910253136A CN 101745115 B CN101745115 B CN 101745115B
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- Prior art keywords
- casein
- acid
- agent
- particle
- solution
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- 239000005018 casein Substances 0.000 title claims abstract description 83
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- 235000021240 caseins Nutrition 0.000 title claims abstract description 83
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- 239000013543 active substance Substances 0.000 claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 230000002378 acidificating effect Effects 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims description 36
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Classifications
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Landscapes
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Abstract
本发明的一个目的在于提供:一种正电性纳米粒子及其制备方法,所述的正电性纳米粒子可以在没有使用表面活性剂或合成聚合物的情况下制备,其大小可以控制,其在酸性条件下是稳定的,并且其中含有活性物质。本发明提供酪蛋白粒子,其中ξ电势为正的。
Description
技术领域
本发明涉及一种用于诸如生命科学或医学诊断之类的领域中的纳米粒子。更具体而言,本发明涉及一种酪蛋白纳米粒子。
背景技术
已经预期细粒材料广泛地用于生物技术。特别是,近年来已经活跃地对由纳米技术的推进而开发出的纳米粒子材料在生物技术或医疗中的应用进行了研究。已经报道了许多研究结果。
纳米粒子在早期在药物递送系统(DDS)领域中受到强烈预期并且极其有希望作为药物或基因的载体。特别是,已经活跃地进行使用聚合物胶束的研究。在大多数情况下,AB-或ABA-型嵌段共聚物由于它们结构的简单性而被使用。聚合物胶束的特征在于其大的药物容量、高的水溶性、高的结构稳定性、非积聚性、小的粒子大小(100nm以下)和功能分离性。鉴于此,进行了意欲靶向靶位和疏水性药物增溶的研究。
近年来,化妆品已经具体化了包括纳米技术在内的各种新技术,因此实现了功能性和使用性的改善以及与其它公司产品的区分。对于化妆品而言,已经需要对皮肤的更明显作用。由于皮肤通常含有作为阻挡层的角质层,因此药物的皮肤透过性低。为了在皮肤上发挥充分的效果,对活性成分的皮肤渗透性的改善是必不可少的。许多成分,即使对皮肤具有高的效果,也难以将其制备成制剂,因为它们具有差的贮存稳定性并且容易引起皮肤刺激。已经寻求意欲改善经皮吸收性和贮存稳定性、减少皮肤刺激等的各种胶囊开发以解决这些问题。目前对于超细乳化或各种原料如脂质体进行了研究(例如,M.Nishida,香料杂志(Fragrance Journal),11月出版,17(2005))。但是,在乳化中使用的表面活性剂出现安全问题。此外,与共价键相比,具有离子配合物的结构形成产生的稳定性差。
预期聚合物材料的使用显著地改善贮存稳定性和体内粒子稳定性。但是,大多数研究使用通过乳液聚合等制备的合成聚合物。尽管与低分子量物质相比,合成聚合物中的毒性得到降低,但是预期其仍具有一定程度的毒性。因此,需要一种更安全的载体。
天然聚合物具有与合成聚合物相同的高结构稳定性,并且具有比合成聚合物的安全性高得多的安全性。因此,天然聚合物同时具有作为DDS载体的优点。但是,与合成聚合物相比,天然聚合物的难点在于制备粒子的方法。可以将喷雾干燥、冷冻干燥和喷射研磨用作用于制备天然聚合物粒子的方法。但是,在大多数情况下,粒子大小是微米大小并且难以控制。
在这样的天然聚合物中,酪蛋白是牛奶中含有的水不溶性蛋白。由于其疏水性部分暴露于外部环境,酪蛋白容易形成聚集体。10至100个酪蛋白分子聚集并且形成约20nm的逊胶束。此外,100至1000个酪蛋白分子聚集并且形成90至150nm的酪蛋白胶束。酪蛋白胶束进一步聚集并且形成约500nm的胶束缔合体。
酪蛋白胶束具有宽的大小分布,并且其在用钠、钾等的盐补充时或在酸性pH放置时聚集。在用于制备酪蛋白逊胶束的常规方法中,使用pH 6.5以上的碱性溶液制备出酪蛋白逊胶束。因此,如果将pH降低至低于酪蛋白等电点的pH值,尽管其是两亲性蛋白,但是发生沉淀和聚集。因此,不能保持这种酪蛋白逊胶束的状态,并且不能制备带正电荷的纳米粒子,结果不能将阴离子化合物结合到纳米粒子或不能保持在粒子表面(日本农业化学协会杂志(Journal of the agricultural chemical society of Japan),第16卷,第9期,1087-1092(1987),日本农业化学协会杂志(Journal of the agriculturalchemical society of Japan),第49卷,第8期,417-424(1975),Journal of DairyResearch,53,547-555(1986)和国际公布WO2007/114262(富士胶片)。
在日本专利公布(Kokai)5-146258A(1993)中,酪蛋白例外地溶解于酸性溶液中。但是,这是意在分馏酪蛋白,因此不意在使用逊胶束。
发明内容
本发明的一个目的在于解决现有技术的上述问题。即,本发明的一个目的在于提供:一种正电性纳米粒子及其制备方法,所述的正电性纳米粒子可以在没有使用表面活性剂或合成聚合物的情况下制备,其大小可以控制,其在酸性条件下是稳定性,并且其中含有活性物质。
作为致力于实现上述目的的深入细致研究的结果,本发明人发现:通过将酪蛋白加入到酸性溶液中,然后将溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值,可以制备其中ξ电势为正的酪蛋白粒子,从而完成了本发明。
本发明提供一种酪蛋白粒子,其中ξ电势为正的。
优选所述酪蛋白粒子的平均粒子直径以粒子大小计为10nm以上至小于300nm。
本发明进一步提供一种酪蛋白粒子,其平均粒子直径为10nm以上至小于300nm,其是由下列步骤(a)和(b)制备的:
(a)将酪蛋白混合到pH为0.5以上至小于pH 7的酸性水介质中的步骤;和
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值的步骤。
优选根据本发明所述的酪蛋白粒子包含在其粒子中和/或在其粒子表面上的活性物质。
优选所述活性物质是离子物质或脂溶性物质。
优选所述活性物质是阴离子物质。
本发明进一步提供根据本发明所述的酪蛋白粒子,其是由下列步骤(a)、(b)和(c)制备的:
(a)将酪蛋白混合到pH为0.5以上至小于pH 7的酸性水介质中的步骤;
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值的步骤;和
(c)将至少一种类型的活性物质加入到在上面的步骤(a)或(b)中得到的溶液中。
优选所述活性物质是化妆品成分、功能食品成分或药物成分。
优选所述化妆品成分是保湿剂、亮肤剂、头发生长刺激剂、头发修复剂、头发生长剂、抗白发剂、染发剂、处理剂、抗老化剂、抗氧化剂、胶原蛋白合成促进剂、抗皱剂、抗粉刺剂、维生素、紫外吸收剂、芳香剂、着色剂、止汗剂、冷却剂、升温剂、黑色素产生抑制剂、黑素细胞活化剂、清洁剂或减轻体重剂;所述功能食品成分是维生素、矿物质、抗氧化剂、抗紧张剂、营养补充剂、氨基酸、类胡罗卜素或水果或蔬菜提取物;并且所述药物成分是头发生长刺激剂、头发修复剂、头发生长剂、抗生素、抗癌剂、抗炎药、抗过敏剂、激素剂、抗血栓剂、免疫抑制剂、用于皮肤疾病的治疗剂、抗真菌剂、核酸剂、麻醉剂、退热剂、止痛剂、止痒剂、抗水肿剂、止咳化痰剂、抗癫痫剂、抗帕金森病剂、镇静催眠药、抗焦虑剂、回苏药、精神神经功能病用药、肌肉松弛药、抗抑郁剂、复合感冒药、自主神经系统剂、解痉药、发汗剂、止汗剂、强心剂、用于心律不齐的治疗剂、抗心律失常药、血管紧张药、血管扩张剂、降血压剂、抗糖尿病剂、用于高脂血症的治疗剂、呼吸兴奋剂、镇咳药、维生素、用于寄生虫皮肤病的药剂、内环境稳定调节剂、多肽、激素、角化不全抑制剂、疫苗或皮肤柔软剂。
本发明进一步提供一种分散材料,其包含在分散介质中的根据本发明所述的酪蛋白粒子。
本发明进一步提供一种用于制备根据本发明所述的酪蛋白粒子的方法,该方法包括将酪蛋白分散在pH低于所述酪蛋白的等电点的溶液中。
本发明进一步提供一种用于制备根据权利要求1所述的酪蛋白粒子的方法,该方法包括下列步骤(a)和(b):
(a)将酪蛋白混合到pH为0.5以上至小于pH 7的酸性水介质中的步骤;和
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值的步骤。
本发明进一步提供一种用于制备根据权利要求4所述的酪蛋白粒子的方法,该方法包括下列步骤(a)、(b)和(c):
(a)将酪蛋白混合到pH 0.5以上至小于pH 7的酸性水介质中的步骤;
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值的步骤;和
(c)将至少一种类型的活性物质加入到在上面的步骤(a)或(b)中得到的溶液中的步骤。
根据本发明,可以制提供一种正电性纳米粒子,其可以在没有使用表面活性剂或合成聚合物的情况下制备,其大小可以控制,其在酸性条件下是稳定的,并且其中含有活性物质。
具体实施方式
以下,将更具体地描述本发明。
本发明的酪蛋白粒子是特征在于其具有正ξ电势的粒子。优选其平均粒子直径按粒子大小计为10nm以上至小于300nm。
本发明中,可以制备具有需要大小的酪蛋白纳米粒子。此外,利用脂溶性活性物质和酪蛋白疏水性部分的相互作用,可以将活性物质结合入酪蛋白纳米粒子中。此外,这些粒子在水溶液中稳定地存在。这样的脂溶性物质的Clog P值优选大于0,更优选为1以上,并且进一步优选为3以上。此外,通过将酪蛋白与离子化合物、离子多糖或不同类型的离子蛋白混合以制备混合粒子,酪蛋白能够在其中含有离子活性物质。即,根据本发明,可以在不使用表面活性剂或合成聚合物的情况下,制备其中含有高度安全的活性物质的纳米粒子。
本发明的酪蛋白纳米粒子具有正电荷,并且其ξ电势为正的。ξ电势起着评价粒子的分散和/或聚集性能、相互作用和表面改性的指示剂的作用。可以由已知方法测量ξ电势。本发明的酪蛋白纳米粒子的ξ电势是正的,并且其优选为3至30(mV)。
本发明的酪蛋白纳米粒子的平均粒子大小通常为10nm以上至小于300nm,优选为10至100nm,并且更优选为10至50nm。
本发明的酪蛋白纳米粒子优选包含至少一种活性物质。活性物质的量没有特别限制。以酪蛋白的重量计,酪蛋白纳米粒子通常包含酪蛋白的0.001至100倍的量的活性物质。
不特别限制本发明中使用的酪蛋白的来源,并且酪蛋白可以衍生自牛奶或豆类。作为这样的酪蛋白,可以使用α-酪蛋白、β-酪蛋白、γ-酪蛋白、κ-酪蛋白以及它们的混合物。也可以使用基因重组酪蛋白。本发明的酪蛋白可以优选以酪蛋白钠盐的形式使用。酪蛋白可以单独使用或组合两种以上类型使用。
用于制备本发明的酪蛋白纳米粒子的方法的一个实例是包括以下步骤的方法:(a)将酪蛋白混合到pH为0.5以上至小于pH 7的酸性水介质中的步骤;和(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点±pH 0.5以上的pH值的步骤。上述步骤(b)还可以通过将在上面的步骤(a)中得到的溶液倾倒入pH为3.5至12的水介质中进行。
当将活性物质结合入粒子中或保持在粒子表面上时,可以将至少一种类型的活性物质加入到在上面的步骤(a)或(b)中得到的溶液中。
适宜时,可以设置酸性水介质的温度,并且该温度通常为0℃至80℃,并且优选为25℃至70℃。本文中使用的这样的酸性水介质的具体实例包括使用有机酸或无机酸的水溶液,所述的有机酸如柠檬酸,抗坏血酸,葡糖酸,羧酸,酒石酸,琥珀酸,乙酸,邻苯二甲酸,三氟乙酸,吗啉代甲磺酸,2-[4-(2-羟基乙基)-1-哌嗪基]乙磺酸,乳酸,丙二酸,马来酸,苹果酸,葡糖醛酸和粘酸;并且所述无机酸如盐酸,高氯酸和碳酸。这样的水溶液的pH是pH 0.5以上至小于pH 7。由于如果pH接近于酪蛋白的等电点,则发生聚集和沉淀,因此优选pH为pH 0.5以上至小于pH 6,并且更优选pH 0.5以上至pH 4以下。作为酸,优选使用有机酸。更优选使用柠檬酸、苹果酸和酒石酸。本发明中,将酪蛋白混合到pH 0.5以上的酸性水介质中时的温度优选为0℃至80℃,并且更优选为10℃至60℃。进一步优选其为20℃至40℃。
在将酪蛋白混合在溶液中之后,作为在搅拌的同时提高酸性水介质溶液的pH的方法,优选向溶液中滴加碱,因为它简单。但是,不特别限制这样方法的类型,只要它满足溶解度、温度和搅拌状态即可。
适宜时,可以确定搅拌速度。可以将其设置在通常100rpm至3,000rpm,并且优选200rpm至2,000rpm。
滴加到溶液中的碱的实例包括但不限于使用无机碱的水溶液和使用有机碱的水溶液,所述无机碱如磷酸钠,磷酸钾,氢氧化钙,氢氧化钠,氢氧化钾和氢氧化镁,并且所述有机碱如三(羟基甲基)氨基甲烷和氨。优选无机碱。更优选氢氧化钠和氢氧化钾。
在将酪蛋白混合到酸性水介质溶液,然后在搅拌的同时提高溶液pH的这样的方法中,在温度升高后的优选pH是等于酪蛋白的等电点±pH 0.5以上,优选±pH 4以下,并且更优选±pH 3以下的pH值。
优选使用注射器进行包括将酪蛋白混合到酸性水介质溶液并且将得到的溶液注射到pH 3.5至12的水介质中的方法,原因在于其操作的简单性。但是,不特别限制该方法,只要其满足注射速率、溶解度、温度和搅拌状态即可。通常,可以以1mL/min至100mL/min的注射速率注射溶液。可以适宜地确定酸性水介质的温度。可以通常将该温度确定在0℃至80℃,并且优选在25℃至70℃。可以适宜地确定水介质的温度。可以通常将该温度确定在0℃至80℃,并且优选在25℃至60℃。可以适宜地设置搅拌速度,并且其可以通常为100rpm至3000rpm,并且优选为200rpm至2000rpm。
可以将有机酸或碱或无机酸或碱的水溶液或缓冲溶液用作本发明中的水介质。
这样的水介质的具体实例包括但不限于:使用有机酸、有机碱、无机酸和无机碱的溶液,所述的有机酸如柠檬酸,抗坏血酸,葡糖酸,羧酸,酒石酸,琥珀酸,乙酸,邻苯二甲酸,三氟乙酸,吗啉代甲磺酸,2-[4-(2-羟基乙基)-1-哌嗪基]乙磺酸,丙二酸,马来酸,苹果酸,葡糖醛酸和粘酸;所述有机碱如三(羟基甲基)氨基甲烷和氨;所述无机酸如盐酸,高氯酸和碳酸;并且所述无机碱如磷酸钠,磷酸钾,氢氧化钙,氢氧化钠,氢氧化钾和氢氧化镁。
本发明中使用的水介质的浓度优选为约5mM至约500mM,并且更优选为约5mM至约200mM。
本发明的酪蛋白粒子优选包含在其粒子中和/或在其粒子表面上的活性物质。本发明中使用的活性物质的类型可以选自例如化妆品成分、功能食品成分和药物成分。本发明的酪蛋白纳米粒子的特征在于,可以将阴离子物质结合于其中或可以保持在表面上。因此,在下述成分中,更优选阴离子化合物。
化妆品成分的实例包括保湿剂、亮肤剂、头发生长刺激剂、头发修复剂、头发生长剂、抗白发剂、抗老化剂、抗氧化剂、胶原蛋白合成促进剂、抗皱剂、抗粉刺剂、维生素、紫外吸收剂、芳香剂、着色剂、止汗剂、冷却剂、升温剂、黑色素产生抑制剂、黑素细胞活化剂、清洁剂和减轻体重剂。
功能食品成分的实例包括维生素、矿物质、抗氧化剂、抗紧张剂、营养补充剂、氨基酸、类胡罗卜素和水果与蔬菜提取物。
药物成分的实例包括头发生长刺激剂、头发修复剂、头发生长剂、抗生素、抗癌剂、抗炎药、抗过敏剂、激素剂、抗血栓剂、免疫抑制剂、用于皮肤疾病的治疗剂、抗真菌剂、核酸剂、麻醉剂、退热剂、止痛剂、止痒剂、抗水肿剂、止咳化痰剂、抗癫痫剂、抗帕金森病剂、镇静催眠药、抗焦虑剂、回苏药、精神神经功能病用药、肌肉松弛药、抗抑郁剂、复合感冒药、自主神经系统剂、解痉药、发汗剂、止汗剂、强心剂、用于心律不齐的治疗剂、抗心律失常药、血管紧张药、血管扩张剂、降血压剂、抗糖尿病剂、用于高脂血症的治疗剂、呼吸兴奋剂、镇咳药、维生素、用于寄生虫皮肤病的药剂、内环境稳定调节剂、多肽、激素、角化不全抑制剂、疫苗和皮肤柔软剂。上述活性物质可以单独使用或组合其两种以上使用。
下面列出本发明中使用的保湿剂的具体实例。但是,在本发明中,保湿剂不限于这些化合物。它包括:透明质酸,神经酰胺,Lipidure,异黄酮,氨基酸和胶原蛋白。上述保湿剂可以单独使用或组合其两种以上使用。
下面列出本发明中使用的亮肤剂的具体实例。但是,在本发明中,亮肤剂不限于这些化合物。它包括:维生素C衍生物,对苯二酚类,熊果苷,Ruciriol和鞣花酸。上述亮肤剂可以单独使用或组合其两种以上类型使用。
下面列出本发明中使用的抗老化剂和抗氧化剂的具体实例。但是,在本发明中,抗老化剂和抗氧化剂不限于这些化合物。它包括:胡萝卜素,维生素A酸,维生素A,维生素C衍生物,激动素,虾青素,维A酸,维生素E及其衍生物,芝麻素,α-硫辛酸,辅酶Q10和类黄酮。上述抗老化剂和抗氧化剂可以单独使用或组合其两种以上类型使用。
下面列出本发明中使用的抗粉刺剂的具体实例。但是,在本发明中,抗粉刺剂不限于这些化合物。它包括:水杨酸,间苯二酚,维生素A酸,那氟沙星,氨基糖苷类抗生素,四环素抗生素和林可霉素抗生素。上述抗粉刺剂可以单独使用或组合其两种以上使用。
下面列出本发明中使用的抗癌剂的具体实例。但是,在本发明中,抗癌剂不限于这些化合物。它包括但不限于:氟化嘧啶基抗代谢药(例如,5-氟尿嘧啶(5FU),替加氟,去氧氟尿苷和卡培他滨);抗生素(例如,丝裂霉素(MMC)和阿霉素(DXR));嘌呤抗代谢药(例如,叶酸抗代谢药(如甲氨蝶呤)和巯嘌呤);维生素A的活性代谢物(例如,抗代谢药(如羟基脲),维A酸和他米巴罗汀);分子靶向药物(例如,曲妥单抗和甲磺酸依马替尼);铂制剂(例如,顺铂(Briplatin)或顺铂(Randa)(CDDP),伯尔定(CBDC),奥沙利铂(Elplat)(Oxa)和奈达铂);植物生物碱药物(例如,托泊替康(Topotecin)或盐酸依立替康(CPT),泰素(PTX),泰索帝(DTX)和依托泊苷);烷基化剂(例如,白消安,环磷酰胺和异环磷酰胺(ifomide));抗雄激素药(例如,比卡鲁胺和氟他胺);雌激素药(例如,磷雌酚,醋酸氯地孕酮和磷酸雌莫司汀);LH-RH药(例如,亮丙瑞林和诺雷德);抗雌激素药(例如,柠檬酸他莫昔芬和柠檬酸托瑞米芬);芳香酶抑制药(例如,盐酸法倔唑,阿那曲唑和依西美坦);黄体激素药(例如,醋酸甲羟孕酮);和BCG。上述抗癌剂可以单独使用或组合其两种以上类型使用。
下面列出本发明中使用的抗过敏剂的具体实例。但是,在本发明中,抗过敏剂不限于这些化合物。它包括:介体抑制剂如色甘酸钠和曲尼司特;组胺H1拮抗剂如富马酸酮替酚和盐酸氮斯汀;血栓素抑制剂如盐酸奥扎格雷;白三烯拮抗剂如普仑司特;和甲磺司特。上述抗过敏剂可以单独使用或组合其两种以上类型使用。
下面列出本发明中使用的免疫抑制剂的具体实例。但是,在本发明中,免疫抑制剂不限于这些化合物。它包括:雷帕霉素,他克莫司,环孢霉素,泼尼松龙,甲基泼尼松龙,麦考酚酸莫酯,硫唑嘌呤和咪唑立宾。上述免疫抑制剂可以单独使用或组合其两种以上类型使用。
本发明中使用的头发生长成分的类型没有特别限制。例如,这样的头发生长成分可以选自:化妆品成分和药物成分。在本发明蛋白纳米粒子中含有的头发生长成分的具体实例包括:甘草次酸或其衍生物;甘草酸或其衍生物;扁柏酚;维生素E或其衍生物;维生素C衍生物;6-苄基氨基嘌呤;烟酰胺;烟酸苄酯;烟酸维生素E;烟酸β-丁氧基酯;异丙基甲基酚;十五烷酸或其衍生物;千金藤碱;非那雄胺;t-黄烷酮;抗氧化剂如类胡罗卜素或激动素;炔雌醇;泛醇;泛酰基乙基醚;米诺地尔或其类似物;卡普氯铵;和腺苷。上述头发生长成分可以单独使用或组合其两种以上类型使用。
本发明中,可以使用与所述活性物质相互作用的离子化合物和添加剂。上述离子化合物和添加剂各自优选具有与活性物质的电荷相反的电荷。相对于活性物质的分子量,离子化合物和添加剂各自优选以0.1至10当量添加。
与所述活性物质相互作用的离子化合物和添加剂可以是阳离子化合物或阴离子化合物。所述化合物优选在其分子中具有疏水性部分和亲水性部分。疏水性部分优选具有共轭结构、环状结构、长链烷基结构和长链亚烷基结构中的一个以上的结构。更优选它具有类固醇结构,或苯甲酰基、联苯或苯基结构。
下面给出与所述活性物质相互作用的上述离子化合物和添加剂的具体实例。但是,离子化合物和添加剂不限于此。特别是,下面列出阴离子化合物:硫酸月桂基酯及其盐,硫酸肉豆蔻基酯及其盐,硫酸鲸蜡基酯及其盐,磷酸月桂基酯及其盐,甲苯磺酸及其盐,甲苯甲酸及其盐,二甲基苯甲酸及其盐,乙酰基苯磺酸及其盐,苯甲酸及其盐,联苯甲酸及其盐,苯甲酰基苯甲酸及其盐,胆酸及其盐,甘氨胆酸及其盐,牛磺胆酸及其盐,鹅去氧胆酸及其盐,猪胆酸及其盐,熊去氧胆酸及其盐,鲤甾醇(cyprinol)及其盐,去氧胆酸及其盐,石胆酸及其盐,猪去氧胆酸及其盐,甘草亭酸及其盐,及甲酸胆固醇及其盐。
在本说明书中,在其中将至少一种活性物质加入到在上面的上述步骤(a)或(b)中得到的溶液的情况下,可以加入通过将所述活性物质溶解于水中或可与水至少10重量%混溶的有机溶剂中而制备的溶液。下面列出在本发明中使用的可与水至少10重量%混溶的有机溶剂的具体实例。但是,在本发明中,有机溶剂不限于这些化合物。优选的实例包括高级醇和多元醇如乙醇、异丙醇、丙二醇、丁二醇、乙二醇和甘油,并且水溶性有机溶剂如丙酮和THF。
本发明中,可以将活性物质以其中含有活性物质的水性脂质体分散液的形式加入。下面列出本发明中使用的用于形成脂质体的脂质的具体实例。但是,在本发明中,脂质不限于这些化合物。它包括:蛋黄卵磷脂,大豆卵磷脂,蛋黄磷脂酰胆碱,二棕榈酰基磷脂酰胆碱和二肉豆蔻酰基磷脂酰胆碱。除了上述化合物外,脂质体还可以包含磷脂酰丝氨酸,磷脂酰乙醇胺和胆固醇。
向本发明的酪蛋白粒子中,可以加入选自环糊精,脂质,不同类型的蛋白,阳离子或阴离子多糖和阳离子或阴离子蛋白中的一种以上的成分。
下面列出在本发明中使用的环糊精的具体实例。但是,在本发明中,环糊精不限于这些化合物。它包括:α-环糊精,β-环糊精,γ-环糊精,2,6-二-O-甲基-α-环糊精,2,6-二-O-甲基-β-环糊精,葡糖苷酸基葡糖基-β-环糊精,七(2,6-二-O-甲基)-β-环糊精,2-羟基乙基-β-环糊精,羟基丙基-β-环糊精,6-O-α-麦芽糖基(maltosyl)-α-环糊精,甲基-β-环糊精,2,3,6-三-O-甲基-β-环糊精和6-O-α-D-葡糖基-α-环糊精。
下面列出在本发明中使用的脂质的具体实例。但是,在本发明中,脂质不限于这些化合物。它包括:磷脂酰胆碱(卵磷脂),磷脂酰乙醇胺,磷脂酰丝氨酸,磷脂酰肌醇,磷脂酰甘油,二磷脂酰甘油,鞘氨醇,神经酰胺,油酸,亚油酸,亚麻酸,棕榈酸,肉豆蔻酸,硬脂酸,大豆油,橄榄油和角鲨烷。
不特别限制在本发明中使用的不同类型的蛋白的种类。优选使用分子量为约10,000至1,000,000的蛋白。不特别限制蛋白的来源。优选使用衍生自人类的蛋白。下面列出蛋白的具体实例。但是,在本发明中,蛋白不限于这些化合物。本文中可以使用的这样不同类型的蛋白的实例包括胶原蛋白,明胶,清蛋白,转铁蛋白,纤维蛋白,纤维蛋白原,球蛋白,丝心蛋白,昆布氨酸(laminin),纤维结合蛋白和玻连蛋白。此外,不特别限制蛋白的来源,并且可以使用牛、猪、鱼和基因重组蛋白中的任何一种。它们中,优选明胶和清蛋白。
本发明中使用的阴离子多糖是具有酸性极性基团如羧基、硫酸基或磷酸基的多糖。下面列出其具体实例。但是,在本发明中,阴离子多糖不限于这些化合物。它包括:硫酸软骨素,硫酸葡聚糖,羧甲基葡聚糖,藻酸,果胶,角叉菜胶,岩藻聚糖,琼脂胶,紫菜聚糖(porphyran),刺梧桐树胶,吉兰胶,黄原胶和透明质酸。
本发明中使用的阳离子多糖是具有碱性极性基团如胺基的多糖。下面列出其具体实例。但是,在本发明中,阳离子多糖不限于这些化合物。它包括:包含葡糖胺(例如,几丁质和壳聚糖)或半乳糖胺作为构成单糖的那些。
本发明中使用的阴离子蛋白包括等电点比生理pH更碱性的蛋白和脂蛋白。下面列出其具体实例。但是,在本发明中,阴离子蛋白不限于这些化合物。它包括:聚谷氨酸,聚天门冬氨酸,细胞色素C,核糖核酸酶,胰蛋白酶原,糜蛋白酶原和α-糜蛋白酶。
本发明中使用的阳离子蛋白包括等电点比生理pH更酸性的蛋白和脂蛋白。下面列出其具体实例。但是,在本发明中,阳离子蛋白不限于这些化合物。它包括:聚赖氨酸,聚精氨酸,组蛋白,鱼精蛋白和卵清蛋白。
本发明的酪蛋白纳米粒子优选在其中包含活性物质。可以将包含活性物质的这样的酪蛋白纳米粒子给予至对于使用受影响的部分。具体而言,可以将本发明的酪蛋白纳米粒子用作药物递送剂。
本发明中,不特别限制药物递送剂的用法。例如,将药物递送剂用作透皮剂、局部治疗剂、口服治疗剂、化妆品、补充剂等。
本发明中,药物递送剂优选包含001%至50重量%的所述蛋白纳米粒子,并且更优选包含0.1%至10重量%的所述蛋白纳米粒子。相对于总量,在药物递送剂中含有的乙醇的量优选为20%以下,并且更优选为10%以下。
本发明中,药物递送剂可以包含添加剂。不特别限制这样的添加剂的类型。这样的添加剂的实例包括保湿剂,软化剂,经皮吸收促进剂,防腐剂,着色剂,芳香剂和pH调节剂。
可以在本发明中使用的保湿剂的具体实例包括但不限于:琼脂,双甘油,二硬脂二铵锂蒙脱石(distearyldimonium hectorite),丁二醇,聚乙二醇,丙二醇,透明质酸钠,己二醇,薏苡仁提取物和凡士林。
可以在本发明中使用的软化剂的具体实例包括但不限于:甘油,矿物油和润肤剂成分(例如异硬脂酸异丙酯,聚异硬脂酸甘油酯,异壬酸异十三烷基酯,异壬酸辛酯,油酸,油酸甘油酯,可可脂,混合脂肪酸甘油三酯,琥珀酸二辛酯,蔗糖乙酸硬脂酸酯,环戊烷硅氧烷,蔗糖二硬脂酸酯,棕榈酸辛酯,羟基硬脂酸辛酯,山嵛酸二十烷酯,蔗糖多山嵛酸酯,聚甲基倍半硅氧烷,肉豆蔻醇,肉豆蔻酸鲸蜡酯,肉豆蔻酸肉豆蔻酯和月桂酸己酯。
可以在本发明中使用的经皮吸收促进剂的具体实例包括但不限于:乙醇,肉豆蔻酸异丙醇,柠檬酸,角鲨烷,油酸,薄荷脑,N-甲基-2-吡咯烷酮,己二酸二乙酯,己二酸二异丙酯,癸二酸二乙酯,癸二酸二异丙酯,棕榈酸异丙酯,油酸异丙酯,油酸辛基十二烷基酯,异硬脂醇,2-辛基十二烷醇,尿素,植物油和动物油。
可以在本发明中使用的防腐剂的具体实例包括但不限于:苯甲酸,苯甲酸钠,羟苯乙酯,山梨酸钾,山梨酸钠,山梨酸,脱氢醋酸钠和羟基苯甲酸甲酯。
可以在本发明中使用的着色剂的具体实例包括但不限于:高岭土,胭脂红,群青蓝,氧化铬和氧化铁。
可以在本发明中使用的芳香剂的具体实例包括但不限于:麝香,金合欢油,茴香油,依兰油,肉桂油,茉莉油,甜橙油,留兰香油,香叶油,百里香油,橙花油,薄荷油,日本扁柏油,茴香油,欧薄荷油,香柠檬油,白柠檬油,薰衣草油,柠檬油,柠檬草油,玫瑰油,玫瑰木油,茴香醛,香叶醇,柠檬醛,灵猫酮,麝香酮,柠檬烯和香草醛。
可以在本发明中使用的pH调节剂的具体实例包括但不限于:柠檬酸钠,乙酸钠,氢氧化钠,氢氧化钾和磷酸。
给予本发明酪蛋白纳米粒子的优选方法包括经皮和跨粘膜(transmucosal)吸收。本发明中可以采用的这样的给予方法的具体实例包括但不限于:外部液体制剂,泥敷剂,涂擦剂,清洁剂,浴用制剂,消毒剂,软膏,凝胶,乳膏,糊剂,泥罨剂,膏药,受伤表面包敷剂,受伤表面包敷纱布,止血剂,粘合剂,粘合带,经皮吸收型粘合带,受伤表面保护剂,气溶胶,洗剂,滋补剂,搽剂,乳剂,混悬液,饱和剂,酊剂,粉剂,泡沫剂,化妆品洗剂,按摩乳,营养霜,面膜,片式外皮制剂,皮肤粘合型化妆产品,唇膏,化妆粉底(makeup base),粉底化妆品,香波,护发素,爽身皂,皂,浴用形式,跨甲剂(transnail agent),鼻粘膜剂,口粘膜剂,直肠粘膜剂,阴道粘膜剂,眼粘膜剂和肺粘膜剂。
根据患者的体重、疾病的状况等,可以适宜地设定本发明酪蛋白纳米粒子的剂量。通常,每次给药给予约20μg至100mg/kg。优选每次给药给予约20μg至50mg/kg。
在下列实施例中更具体地描述本发明。但是,这些实施例不意在限制本发明的范围。
实施例
实施例1:
将100mg的酪蛋白(衍生自牛奶,由Wako Pure Chemical Industries,Ltd.制造;等电点:4.3)混合到10ml的50mM柠檬酸溶液(pH 1.9)中。将NaOH加入到混合溶液中,使得pH能够稳定地保持在pH 3。使用光散射光度计(Nano-ZS;由Malvem Instruments Ltd.制造)测量上述粒子的平均粒子直径。结果,测得其为22nm,并且测得ξ电势为16mV(表1)。此外,证实粒子在4℃稳定地分散了10天。
实施例2和3
以与实施例1相同的方式制备酪蛋白纳米粒子,不同之后在于,将加入NaOH后的最终pH设置在pH 2.1(实施例2)或pH 3.9(实施例3)。然后,测量粒子大小(nm)和ξ电势(mV)。结果示于表1中。
比较例1至5
以与实施例1相同的方式制备酪蛋白纳米粒子,不同之后在于,将加入NaOH后的最终pH设置在pH 5.0(比较例1),pH 6.5(比较例2),pH 7.0(比较例3),pH 7.9(比较例4)或pH 10.0(比较例5)。然后,测量粒子大小(nm)和ξ电势(mV)。结果示于表1中。
表1:
实施例2 | 实施例1 | 实施例3 | 比较例1 | 比较例2 | 比较例3 | 比较例4 | 比较例5 | |
pH | 2.1 | 3.0 | 3.9 | 5.0 | 6.5 | 7.0 | 7.9 | 10.0 |
粒子大小(nm) | 46 | 22 | 1110 | 184 | 22 | 21 | 19 | 18 |
ξ电势(mV) | 23 | 16 | 4 | -15 | -14 | -15 | -15 | -15 |
备注 | 本发明 | 本发明 | 本发明 | 比较例 | 比较例 | 比较例 | 比较例 | 比较例 |
在表1中所示的比较例1至5中,粒子具有负ξ电势,因此不能实现本发明的目的。尽管然后将比较例1至5的粒子的pH转化至pH 3,但是它们没有被再分散。因而,清楚的是,当利用酪蛋白纳米粒子的阳离子性质时,需要在酸性范围内制备纳米粒子。在实施例3中,发生聚集和/或沉淀。
实施例4:
使用苹果酸代替实施例1中的柠檬酸制备酪蛋白纳米粒子,并且得到pH 2.4的分散溶液。使用光散射光度计(Nano-ZS;由Malvem Instruments Ltd.制造)测量上述粒子的平均粒子直径。结果,测得其为38nm,并且测得ξ电势为24mV。此外,证实粒子在4℃稳定地分散了10天。
实施例5:
使用酒石酸代替实施例1中的柠檬酸制备酪蛋白纳米粒子,并且得到pH 2.4的分散溶液。使用光散射光度计(Nano-ZS;由Malvem Instruments Ltd.制造)测量上述粒子的平均粒子直径。结果,测得其为11nm,并且测得ξ电势为23mV。此外,证实粒子在4℃稳定地分散了10天。
实施例6:
以与实施例1中相同的方式制备酪蛋白纳米粒子,不同之处在于将实施例1中使用的酪蛋白的量改变为200mg。得到pH 2.6的分散溶液。使用光散射光度计(Nano-ZS;由Malvem Instruments Ltd.制造)测量上述粒子的平均粒子直径。结果,测得其为26nm,并且测得ξ电势为18mV。
比较例6
将100mg的酪蛋白(衍生自牛奶,由Wako Pure Chemical Industries,Ltd.制造)混合到10ml的50mM磷酸缓冲液(pH 10)中。在搅拌的同时,将盐酸加入到混合溶液中,使得pH被调节至pH 4。结果溶液变浑浊并且聚集。
比较例7
将100mg的酪蛋白(衍生自牛奶,由Wako Pure Chemical Industries,Ltd.制造)混合到10ml的50mM磷酸缓冲液(pH 10)中。在搅拌的同时,将盐酸加入到混合溶液中,使得pH被调节至pH 2。结果溶液变浑浊并且聚集。
Claims (8)
1.一种酪蛋白粒子,其中ξ电势为正的,其平均粒子直径为10nm以上至小于300nm,其是通过包括下列步骤(a)和(b)的方法制备的:
(a)将酪蛋白混合到pH为0.5以上至小于pH7的酸性水介质中的步骤;和
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点-pH0.5以上的pH值的步骤。
2.根据权利要求1所述的酪蛋白粒子,其包含在其粒子中和/或在其粒子表面上的活性物质,其中所述活性物质是离子物质或脂溶性物质。
3.根据权利要求2所述的酪蛋白粒子,其中所述活性物质是阴离子物质。
4.根据权利要求2所述的酪蛋白粒子,其中所述活性物质是化妆品成分。
5.根据权利要求4所述的酪蛋白粒子,其中所述化妆品成分是选自下列各项中的至少一种化妆品成分:保湿剂、亮肤剂、头发生长刺激剂、头发生长剂、抗白发剂、染发剂、抗老化剂、抗氧化剂、胶原蛋白合成促进剂、抗皱剂、抗粉刺剂或维生素。
6.一种分散材料,其包含在分散介质中的根据权利要求1至5中任一项所述的酪蛋白粒子。
7.一种用于制备根据权利要求1所述的酪蛋白粒子的方法,该方法包括下列步骤(a)和(b):
(a)将酪蛋白混合到pH0.5以上至小于pH7的酸性水介质中的步骤;和
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点-pH0.5以上的pH值的步骤。
8.一种用于制备根据权利要求2至5中任一项所述的酪蛋白粒子的方法,该方法包括下列步骤(a)、(b)和(c):
(a)将酪蛋白混合到pH0.5以上至小于pH7的酸性水介质中的步骤;
(b)在搅拌在上面的步骤(a)中得到的溶液的同时,将所述溶液的pH增加到离开所述酪蛋白的等电点-pH0.5以上的pH值的步骤;和
(c)将至少一种类型的活性物质加入到在上面的步骤(a)或(b)中得到的溶液中的步骤。
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