功能性磁性纳米微粒的制备、表征以及川芎嗪载药的初步应用
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摘要
研究背景：
随着时代的进步,环境的恶化,疾病谱的发展变化,疾病的诊治在现代医学领域里受到了挑战,而中医药在现代临床治疗中逐渐又焕发出新的魅力和迎来新的机遇。中药或中药制剂现已广泛应用于临床,但中药或中药制剂存在着生物利用度低、有效浓度低、起效缓、半衰期短、穿血脑屏障能力差等缺点,严重阻碍其临床疗效的发挥,亦制约了现代中医药的发展。近年来,纳米载药技术已逐渐应用于中医药领域,如中药纳米化、纳米包合技术、聚合物纳米粒载体技术、脂质体纳米粒、固体分散技术等,但仍处于起步阶段,存在许多亟待解决的问题,如纳米中药的药效不确定性以及可能的毒副作用,纳米中药的有效成分和稳定性难以控制等。诸多中药的有效成分为难溶性物质,口服给药吸收差、生物利用度低,故找出合适的方法来提高中药难溶性有效成分的溶解度,改善其生物利用度,是当前中医药工作者面临的主要挑战之一。将中医药复方的有效成分嫁接到磁性纳米微粒上,使其成为生物利用度高、有控缓性能、靶向性的新的药物剂型的研究尚未报道,本课题将从川芎嗪(复方正天丸君药川芎的主要药物成分)的载药开始研究,逐步寻找中医药复方完美的给药途径。磁性纳米材料与中药单体川芎嗪结合组成复合纳米微球,能很好地改善生物利用度低、起效缓、血药浓度低、半衰期短、穿血脑屏障能力差等缺点；磁性纳米粒子具有超顺磁性、小尺寸效应、表面效应、量子隧道效应、磁响应性、生物相容性和生物降解性、功能基团等特性,由于其独特的优势在生命科学领域显示了良好的应用前景。
研究目的：
通过对磁性纳米微粒的制备、表征以及对牛血清蛋白吸附性能测定的综合评价来寻找一种相对有很好载药潜质的并具有超顺磁性、靶向性和生物相容性等优异特性的磁性纳米微粒作为中药的载体,以便更好地改善中药或中药制剂的生物利用度低、起效缓、血药浓度低、半衰期短、穿血脑屏障能力差等缺点。为川芎嗪寻找一种新的剂型,为偏头痛治疗寻找新的良方。
研究方法：
1.磁性纳米粒子铁酸钴镍(Ni0.5Co0.5Fe2O4)的制备、表征及其牛血清蛋白(BSA)吸附性能的测定。
1.1采用共沉淀法制备Ni0.5Co0.5Fe2O4(NCFO)纳米粉体,以NiCl2·6H2O(98%), CoC4H6O4·4H2O(99.5%)和Fe(NO3)3·9H2O(98.5%)为前驱物,将前驱物按所需的化学计量比分别溶于去离子水中搅拌,然后再将三者混合,将混匀的混合溶液加热至800C恒温,搅拌2小时(h)后,一边搅拌一边往溶液中加入氨水,直到溶液的酸碱度(pH)值约等于8.5,停止加入氨水,然后再搅拌大约30分钟后,再加入酒精球磨24h,最后放置干燥箱中干燥后得到所需的前驱体粉体。将得到的粉体样品在通氮气(N2)气氛下并不同温度(5500C到10500C)下退火。
1.2将制备所得的纳米粉体Ni0.5Co0.5Fe2O4样品采用X射线衍射(XRD,(CuKα：λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对合成的样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子Ni0.5Co0.5Fe2O4,然后将其置于铜网上晾干,用透射电子显微镜(TEM,HITACHIH7650日本)观察磁性纳米粒子Ni0.5Co0.5Fe2O4的形态和粒径。
1.3磁性纳米粒子Ni0.5ECo0.5Fe2O4对牛血清蛋白的吸附性能的测定。具体方法如下：将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中配制成pH等于7.4、浓度为1.000mg/ml的溶液,将适量的磁性纳米粒子Ni0.5Co0.5Fe2O4溶于配制好的牛血清蛋白溶液中,将此混合溶液在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米粒子Ni0.5Co0.5Fe2O4对牛血清蛋白的吸附性能。
2.磁性纳米粒子镍锰酸镧(La2NiMnO6)的制备、表征及其牛血清蛋白(BSA)的吸附性能的测定。
2.1采用化学共沉淀法制备La2NiMnO6纳米粉体。具体实验步骤如下：以硝酸镧(La(NO3)3·5H2O(99.5%)),醋酸镍(Ni(CH3COO)2·4H2O(98%))和醋酸锰(Mn(CH3COO)4·4H2O(99%))为前驱物,将前驱物按所需的化学计量比分别溶于去离子水中搅拌,然后再将三者混合,将混合溶液加热至80℃恒温,搅拌2小时后一边搅拌一边往溶液中加入氨水,直到溶液的pH值约等于8.5为止。搅拌大约30分钟后,再加入酒精球磨24小时,然后放置干燥箱中干燥后得到所需的前驱体粉体。将得到的粉体在氮气(N2)气氛下不同温度(750℃,850℃,950℃,1050℃)下退火。
2.2退火获得的磁性纳米粉体La2NiMnO6的表征。退火获得粉体样品的采用X射线衍射(XRD,(CuKα:λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对获得粉体样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子La2NiMnO6然后置于铜网上晾干,用透射电子显微镜(TEM, HITACHIH7650日本产)观察磁性纳米粒子La2NiMnO6的形态和粒径。
2.3磁性纳米粒子La2NiMnO6对牛血清蛋白的吸附性能的测定。具体实验步骤如下：将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中配制成pH等于7.4、浓度为1.000mg/ml的溶液,将适量的磁性纳米粒子La2NiMnO6溶于已配制好的牛血清蛋白溶液中,将此混合溶液在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米微粒La2NiMnO6对牛血清蛋白的吸附性能。
3.磁性纳米粒子铁酸锌镍(Ni0.5Zn0.5Fe2O4)的制备、表征及其牛血清蛋白(BSA)的吸附性能的测定。
3.1采用共沉淀法制备Ni0.5Zn0.5Fe2O4纳米粉体,以醋酸镍(Ni(CH3COO)2·4H2O(98%)),醋酸锌(Zn(CH3COO)2·2H2O(99%)),和硝酸铁(Fe(NO3)3·9H2O(99%))为前驱物,将Ni(CH3COO)2·4H2O(98%)和Zn(CH3COO)2·2H2O(99%)溶于36%的乙酸中组成混合溶液甲,将Fe(NO3)3·9H2O(99%)溶于去离子水组成混合液乙,然后再将甲混合液逐滴加入乙溶液中,边滴边用磁力搅拌,同时加热至80℃后恒温,继续将混合溶液磁力搅拌2小时后,一边搅拌一边往混合溶液中加入氨水,直到溶液的pH值约等于8.5为止。然后静止沉淀、自然冷却后过滤,然后放置干燥箱中干燥24小时,取得干燥混合物再加入酒精球磨后预烧,之后再加入酒精球磨后在通氮气(N2)气氛下并不同温度(400℃,500℃,600℃,700℃,800℃)下退火。
3.2退火获得的磁性纳米粉体Ni0.5Zn0.5Fe2O4的表征。采用X射线衍射(XRD,(CuKα:λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对退火所获得的磁性纳米粉体Ni0.5Zri0.5Fe2O4的样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子Ni0.5Zn0.5Fe2O4然后将其置于铜网上晾干,用透射电子显微镜(TEM,HITACHIH7650日本)观察磁性纳米粒子Ni0.5Zn0.5Fe2O4的形态和粒径。
3.3采用紫外线分光光度计(UV2401pc)来分析磁性纳米粒子Ni0.5Zn0.5Fe2O4对牛血清蛋白的吸附性能,将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中分别配制成pH等于7.0和3.0浓度为1.000mg/ml的牛血清蛋白溶液,将适量的磁性纳米粒子Ni0.5Zn0.5Fe2O4溶于牛血清蛋白溶液中,将此混合物在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米粒子Ni0.5Zn0.5Fe3O4对牛血清蛋白在不同酸碱度的环境下(pH=7.0,3.0)的吸附性能。然后选择吸附力强的pH环境的磁性纳米粒子Ni0.5Zn0.5Fe2O4样品,在常温下超声搅拌不同的时间(30-120min),来观察搅拌时间对牛血清蛋白的吸附性能的影响。
4.吐温-80修饰的荧光标记的磁性纳米粒子与川芎嗪组成的复合纳米微粒的制备、表征及其性能的测定。
4.1采用共沉淀法制备荧光粉体LaPO4:Eu,将称量好的Y2O3, La2O3和Eu203溶于l0ml浓硝酸+10m1去离子水混合溶液中,然后加热至80℃C,30分钟后全溶,随后将(NH4)2HPO4直接加入,充分搅拌溶解后,调节PH值为9.5,再继续搅拌2小时。过滤后用去离子水反复冲洗,然后放在干燥箱中烘24小时,研磨,预烧。最后在不同温度下退火。
4.2复合纳米微粒的制备。具体实验步骤如下：用2毫升(m1)浓度为25%的牛血清白蛋白加入20mmg四甲基吡嗪(川芎嗪TMP),再加入30mg磁性纳米粒子镍锰酸镧(La2NiMnO6退火温度为850摄氏度的样品)、15mg荧光粉体掺铕磷酸镧(LaPO4:Eu退火温度为800摄氏度的样品)充分搅拌,再将搅拌均匀的混合物加入40m1的吐温80中,在常温下超声乳化20分钟(min),再取100m1吐温80加热到120摄氏度,将加热的吐温80置于加热器上维持恒温,将超声乳化后的混悬液以100滴／分钟的速度滴入振荡的预热的吐温80中,加热振荡维持恒温10min；然后用冰冷却至25℃,用无水乙醚洗涤3次,每次用无水乙醚50m1,然后3000转／分钟(R/min)离心15min,离心后的复合物待自然蒸发干燥后,4℃C下保存备用。
4.3将制备好的复合纳米微粒采用XRD(CuKα:A=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02。／2s,衍射角从10。-80。。在30kOe的磁场中,采用VSM(MPMS,美国QUANTUM DESIGN公司)对合成的复合纳米微球样品进行了磁性能的测试。用无水乙醇分散复合纳米粒子,然后置于铜网上,高倍电子显微镜观察复合纳米粒的形态和粒径。
4.4使用荧光光度计测量复合纳米微粒中川芎嗪(TMP)的含量,将复合纳米微粒样品按1:5(mg/ml)加入浓度为5%的稀盐酸溶液中,在超声混匀后8℃下静置24小时,然后2000r/min离心l0min,取上清液,用荧光光度计测量川芎嗪含量。
4.5复合磁性纳米微粒中川芎嗪(TMP)体外释放测定；精密称取制备好的复合纳米微粒,加入适量磷酸盐缓冲溶液(含0.2%叠氮钠作为抑菌剂,0.1%Tween-80作为润湿剂)为释放介质,置于恒温水浴摇床中,在100r/min振荡速度、37℃条件下进行复合纳米微粒中川芎嗪的体外释放速度测定。分别在设定时间(0.5h、1h、2h、5h、10h、15h、20h、25h、30h、35h、40h)取出,于15000r/min离心15min,吸出上清液后,加入等量新鲜的释放介质。采用荧光光度计测量川芎嗪的含量。计算川芎嗪在磷酸盐缓冲溶液(pH7.4,37℃)中的累积释药百分率,以累积释药百分率对时间作图。
4.6复合磁性纳米微粒的细胞毒性测定,采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力影响。
4.7数据分析：实验结果计量资料用均数±标准差(x±s)表示,细胞毒性实验中,实验组与对照组比较采用单因素方差分析,用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义；累积释药百分率散点图采用曲线拟合法拟合。
结果：
1.通过化学共沉淀法成功制备磁性纳米粒子Ni0.5Co0.5Fe2O4(NCFO)。X射线衍射(XRD)分析显示各个不同退火的磁性纳米微粒NCFO样品全都为典型的单相立方尖晶石结构,随着退火温度从550上升到950℃C,磁化强度(Ms)从35.95增加到67.19emu/g。当退火温度进一步升高至1050℃时,Ms有所减小。NCFO纳米粒子的饱和磁化强度和晶粒尺寸最初均随退火温度的上升而增加,后又随着退火温度的上升而有所减小。用透射电子显微镜(TEM, HITACHIH7650)来测定磁性纳米粒子NCFO的粒径,所测得的平均粒径约为50nm。磁性纳米粒子NCFO展现出了对牛血清蛋白有良好的吸附性能,当退火温度处于750℃其粒径33.3nm的磁性纳米微粒NCFO样品显示最强的吸附力为71(mg/g)左右。
2.磁性纳米粒子La2NiMnO6(LNMO)采用化学共沉淀法成功制备。LNMO的晶粒尺寸极大地受到退火温度的影响。随着退火温度从750℃升高到1050℃,它的平均晶粒尺寸从33.9nm增加到了39.6nm,另一方面,矫顽力随着退火温度的升高先增加,950℃退火,平均晶粒尺寸为37.9nm样品的矫顽力达到最大42.30e,然后随着退火温度的继续升高,矫顽力随之减少。该LNMO纳米微粒表现出了对牛血清白蛋白良好的吸附性能,磁性纳米粒子LNMO在850℃温度下退火样品的BSA吸附能力最强,大约为219.6mg/g。在此情况下,吸附后BSA溶液的体积增加了约3m1。
3.采用化学共沉淀法成功制备了磁性纳米粒子Ni0.5Zn0.5Fe2O4(NZFO),通过XRD技术来表征,NZFO粉末形成单一的立方尖晶石结构,并且没有其它杂相的产生。退火温度对磁性纳米粉体Ni0.5Zn0.5Fe2O4晶粒尺寸与磁性能都有一定的影响。随着退火温度的增加,饱和磁化强度增强。在牛血清蛋白溶液的pH=7.0的环境下,退火温度为600℃时的磁性纳米粒子Ni0.5Zn0.5Fe2O4样品对牛血清蛋白的吸附性能为最强,其吸附值约为38.35mg/g。
4.经过吐温-80修饰、掺铕磷酸镧(LaP04:Eu)荧光粉体标记的复合磁性纳米微粒(川芎嗪单体、牛血清白蛋白、La(Ni0.5Mn0.5)O3(LNMO-850)、LaPO4:Eu-800、吐温80共同组成的复合物)采用高温使蛋白质固化的原理成功制备。经高倍电子显微镜观察复合纳米微球的粒径约为0.5um,并且测得其有较高的载药量和包封率。通过对复合磁性纳米微粒的体外释放的测定发现,川芎嗪经吐温80修饰和磁性纳米粒子包封后呈现出明显的缓释性能。数据通过曲线拟合,拟合度为0.56476,结果显示复合磁性纳米微粒的半衰期约为25小时。采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力的影响,经统计分析,纳米药物组与对照组对HepG2人肝癌细胞株增殖活力比较差异无统计学意义(F=3.153P=0.150),结果显示,在一定浓度范围内复合磁性纳米微粒是安全的。
结论：
通过对铁酸钴镍(Ni0.5Co0.5Fe2O4)、镍锰酸镧(La2NiMnO6)、铁酸锌镍(Ni0.5Zn0.5Fe2O4)等三种磁性纳米粒子的制备、表征及其牛血清蛋白吸附性能的测定的综合评价后,选择了在氮气气氛中退火温度为850℃的镍锰酸镧作为制备复合纳米微粒的备选磁性纳米粒子样品。经吐温80修饰、掺铕磷酸镧(LaPO4:Eu)荧光粉体标记的复合磁性纳米微粒(川芎嗪单体、牛血清白蛋白、La2NiMnO6(LNMO-850)、LaP04:Eu-800、吐温80共同组成的复合物)采用高温固化的原理成功制备。实验结果表明川芎嗪在复合磁性纳米微粒中有较高的载药量,且经吐温80修饰、磁性纳米粒子包封后呈现出明显的缓释性能。采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力的影响,结果显示在适当的浓度范围复合磁性纳米微粒是安全的。本研究利用磁性纳米粒子为载体成功载药中药单体川芎嗪,为川芎嗪提供了一种新的剂型,为中药复方载药打下了坚实的基础,为偏头痛的治疗带来了新的曙光。
Background:
With the progress of time, environmental degradation and the development of spectrum of disease, diagnosis and treatment of diseases in the field of modern medicine has been challenged. However, the modern clinical medicine in the treatment is gradually fulled of new charm and new opportunities.Nowadays, traditional Chinese medications and its processed drugs are widely used in clinical practice,but they are same shortcomings such as low bioavailability, low effective concentration, slow onset, short half-life, and penetrating poorly blood-brain barrier,which not only seriously hinder drugs effectiveness, but also restrict the development of modern medication.
In recent years, drug-loaded technology of nanoparticles used in the medical field, for example, nano-medicine, nano-inclusion technique, polymer nano-particles carrier technology, liposome nano-particles, solid dispersion technology and other fields, these are still in early stage and many problems need to be solved,including uncertainty efficacy of nano-medicine and possible side effects, difficultly control on active ingredients and instability of nano-medicine.Because of many of poorly soluble active ingredients of traditional Chinese medication, poor oral absorption and low bioavailability,therefore, it is necessary to find the right way to improve the solubility of poorly soluble active ingredients of traditional Chinese medication and its bioavailability, which is one of the main challenges faced by medical workers.
The research that the active ingredients of Chinese herbal compound are grafted onto magnetic nano-particles in order to enhance bioavailability and the performance of controlled slowly drugs and the formulation of targeted new drug has not been reported,This paper will start with one of the major drug with ligustrazine (TMP)of Zheng Tian Pill,gradually finding more perfect route of administration of traditional Chinese medication.Integrating magnetic nano-materials to medicine TMP monomers together form a composite nano-spheres that can well improve defects such as low bioavailability, slow onset, low plasma concentration, short half-life, the poor ability to wear blood-brain barrier.As magnetic nano-particles have super-paramagnetic, small size effect, surface effect, quantum tunneling effect,magnetic response,biocompatibility and biodegradability, functional groups and other characteristics, its unique advantages in the field of life sciences show a promising prospect.
Objective:
Based on the preparation, characterization of magnetic nanoparticles and the comprehensive evaluation for the determination of bovine serum albumin adsorption performance to look for a relatively good magnetic nano-particles with super-paramagnetic, targeting and biocompatibility excellent properties as a carrier of traditional Chinese medicine in order to better improve the low bioavailability of traditional Chinese medication or its processed drugs, slow efficacious, low plasma concentration,the short half-life,badly wearing blood-brain barrier,etc,which can help look for a new dosage of TMP and a new recipe of the treatment of migraine headache.
Methods:
1.The preparation and characterization of nickel Cobalt ferrite(Ni0.5Co0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
1.1The Nio.5Co0.5Fe2O4nano-composites were synthesized by co-precipitation, using NiCl2·6H2O (98%), CoC4H6O4·4H2O(99.5%), and Fe(NO3)3·9H2O (98.5%) as starting raw materials without further purification. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5. After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in N2atmosphere for2h at different temperatures in the range of550～1050℃.
1.2The crystalline phase of Ni0.5Co0.5Fe2O4nano-composites was identified by the x-ray diffraction (XRD) technique. X-ray diffraction gram of all the samples from10°to80°at a scanning step of0.02°/2s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (1=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM) at a room temperature under a maximum field of30kOe.
1.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>98%)1.000mg/ml before the adsorption and for each measurement,3.00to15.00mg nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the aqueous BSA solution for1h at room temperature.
2.The preparation and characterization of Lanthanum-nickel-manganate(La2NiMnO6) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
2.1The La2NiMnO6(LNMO) nano-composites were synthesized by co-precipitation, using La(NO3)3-5H2O(99.5%), Ni(CH3COO)24H2O (98%) and Mn(CH3COO)4-4H2O(99%) as starting raw materials. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h,aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5.After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of750～1050℃.
2.2The crystalline phase of LNMO nano-composites was identified by the X-ray diffraction (XRD)technique. X-ray diffractogram of all the samples from10°to80°at a scanning step of0.02°/s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (X,=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA)at a room temperature under a maximum field of30kOe. The structural defects in LNMO materials were investigated using J EOL4000EX high resolution transmission electron microscope (HR-TEM) operated at400kV.
2.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,La(Ni0.5Mn0.5)O3nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution for1h at room temperature,which put in static precipitation condition after12h to be measured.
3.The preparation and characterization of nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
3.1The Nio.5Zno.5Fe204nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. The Ni0.5Zn0.5Fe2O4nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. Before both solutions were dissolved in acetic acid (CH3COOH36%), and the latter were dissolved in deionized water, then the former mixed solutions drip into the latter mixed solutions dropwise and stirred meanwhile in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the last mixed solutions, until brown suspension took shape at pH-8.5. The obtained suspension were filtrated and dried in a cabinet dryer at80℃overnight to obtain the precursor samples, which was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough after presintering. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of400-800℃.
3.2Characterizations of Ni0.5Zn0.5Fe2O4nano-particles. The structure and morphologies of the resultant nano-particles were determined by X-ray diffraction (XRD) and transmission electron microscopy (TEM,HITACHIH-7650). The magnetic properties were measured by using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.
3.3The adsorption of bovine serum albumin (BSA (V)) protein on the Nio.5Zno.5Fe204nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.0and3.0contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,6.00to13.00mg of the Ni0.5Zn0.5Fe2O4nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution (pH about7.0and3.0) for1h at room temperature, and which the strongest adsorption was stirred ultrasonically in the different time, which put in Static precipitation condition after12h to be measured.
4. To measurement on the preparation, characterization and properties composited nano-particles combining modified tween80fluorescently was labeled magnetic nano-particles of and TMP
4.1The LaYPO4:Eu powder were synthesized by co-precipitation, using Y2O3, La2O3and Eu2O3as starting raw materials were dissolved in mixed solution of HNO3(69%,10ml) and deionized water(10ml) in required stoichiometric proportions. The mixed solutions were heated to80℃. After2h,(NH4)2HPO4solution was added to the container, until the suspension took shape at pH=9.5. After stirring for about2h the suspension was filtered and then rinsed repeatedly with deionized water. Afterwards dried in a cabinet dryer at80℃overnight to obtain the precursor samples. After grinding and presintering, the dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of500～800℃.
4.220mg Ligustrazine(TMP) add in the bovine serum albumin (BSA(v)25%,2ml).30mg La(Ni0.5Mn0.5)O3(LNMO950℃) nano-particles and15mg LaYPO4:Eu powders join in the mixed solution,then string fully. Compound of after string join in40ml Polysorbate,then the admixture was stirred ultrasonically in the BSA solution for10min at room temperature.100ml heated to120℃to maintain a constant temperature in heater.after the suspensions were ultrasonic stirred dropping in Polysorbate of with maintaining a constant temperature100drop/min speed and vibrate10min.Then was cooled to25degree with ice. With deionized water washing three times, each time with deionized water50ml,centrifuge15min with3000R/min,After centrifugal composite nano-particles to natural evaporation drying,4degrees to save backup.
4.3The structure and morphologies of the resultant nanoparticles were determined by X-ray diffraction (XRD) of all the samples from10°to80°at a scanning step of0.02°/2s was recorded.The magnetic properties were measured by using a vibrating sample magnetometer (VSM, quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.Dispersed composite nano-particles with ethanol, and then placed in a copper-line, high-powered electron microscope composite nano-particle morphology and particle size.
4.4First,use fluorometer to measure the content of TMP in composite nano-particles then,samples of composite nano-particles by1:5(mg/ml) was added at a concentration of5%of dilute hydrochloric acid solution and allowed to stand at8℃after mixing ultrasound for24hours.Last,2000r/min centrifugal10min, the supernatant was used to measure the TMP content by fluorescence photometer.
4.5Composite magnetic nano-particles TMP (TMP) in vitro release assay;prepared composite nano-particles by accurately weighed was added the appropriate amount of phosphate buffer solution (containing0.2%sodium azide as an antimicrobial agent,0.1%Tween-80as a wetting agent) using for the release of the medium,which was placed in a water bath shaker at100r/min shaking speed,37℃condition in order to determinate the vitro release rate of composite nano-particles TMP. Respectively, in the set time (0.5,1,2,5,10,15,20,25,30,35, and40h) removed by centrifugation at15000r/min15min, After aspiration of the supernatant was added an equal amount of fresh release medium. The content of TMP using fluorescence spectrometer measurements. TMP in phosphate buffer solution is calculated(pH7.4,37℃) cumulative release percentage and cumulative release percentage plotted against time.
4.6Cytotoxicity determination of the composite magnetic nano-particles, detecting the influence of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay.
4.7Data analysis:The results of measurement data with mean±standard deviation (x±s) is that the cytotoxicity experiments, the experimental and control groups were compared using ANOVA with SPSS13.0statistical software for analysis, P<0.05set for the difference was statistically significant, using curve fitting fit scatter plot.
Result:
1. The Ni0.5Co0.5Fe2O4(NCFO) nano-particles have been successfully prepared by the chemical co-precipitation process. XRD analysis showed that the each of sample magnetic nano-particles NCFO were all the typical single-phase cubic spinel structure,with the annealing temperature rises from550to950℃, magnetization Ms from35.95to67.19emu/g, when the annealing temperature was further increased to1050℃, Ms decreased. NCFO nano-particles of Grain size and saturation magnetization were initially increased with increasing annealing temperature, and then reduced with increasing annealing temperature and determined the average particle diameter of magnetic nano-particles in NCFO using transmission electron microscope ending with The average particle diameter of about50nm.NCFO magnetic nano-particles showed a bovine serum albumin had good adsorption properties, when the annealing temperature was750℃particle size of magnetic nano-particles NCFO33.3nm revealed the strongest suction force is71(mg/g) or so.
2. La2NiMnO6(LNMO) nano-particles have been successfully prepared by the by the chemical co-precipitation process.The grain size of the LNMO nano-particles are largely influenced by annealing temperature.As the annealing temperature increases from750to1050℃, the average grain size increases from about33.9to39.6nm, On the other hand, the coercivity initially increases, reaching a maximum value of42.3Oe when the average grain size was about37.9nm at950℃, and then reduces. The LNMO nano-particles showed good adsorption performance in bovine serum albumin protein, and the preliminary optimized adsorption was obtained for the LNMO nano-particles annealed at850℃.These LNMO nano-particles were a potential carrier for large biomolecules, which will be widely used in the biomedical field.
3.Successfully prepared by chemical magnetic nano-particles Ni0.5Zn0.5Fe2O4(ZNFO) by co-precipitation method,Characterized by XRD technique, NZFO powder formed a single cubic spinel structure, and do not produce other miscellaneous phase.Annealing temperature on the grain size of magnetic nanoparticles and magnetic powder NZFO had some impacts. With increasing annealing temperature, the saturation magnetization was enhanced. When bovine serum albumin solution at a pH of7.0environment, the annealing temperature was600℃, magnetic nano-particles NZFO samples had the strongest adsorption performance of bovine serum protein, that is, the adsorption value of about38.35mg/g.
4. After modification of Tween80, europium-doped lanthanum phosphate (LaPO4: Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin V,La(Ni0.5Mn0.5)O3(LNMO-850), LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein. The size of composite nano-pheres was about0.5μm and obtain higher drug loading and encapsulation efficiency by high-powered electron microscope. In vitro by measuring the release of the composite magnetic nano-particles and found that TMP modified by Tween80and magnetic nano-particle entrapment showed a significant slow-release properties. Data by curve fitting, the fit is0.56476. The results showed that the half-life of the composite magnetic nano-particles was about25h. Composite magnetic nano-particles to detect the impact of human hepatoma cell line HepG2proliferation activity by MTT assay showed that the composite magnetic nano-particles are safe. After statistical analysis, both nano-drug group and the control group showed no significant difference in HepG2human hepatoma cell line proliferation activity.
Conclusion:
the preparation and characterization of three magnetic nano-particles such as Iron-nickel-cobalt(Ni0.5Co0.5Fe2O4), nickel lanthanum manganite (La2NiMnO6), nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4), and the measurement and evaluation on bovine serum albumin the of adsorption performance. Alternatively magnetic nano-particles selected samples in a nitrogen atmosphere, lanthanum-nickel-manganese annealing temperature of850℃as the preparation of the composite nanoparticles.After modification of Tween80, europium-doped lanthanum phosphate (LaPO4:Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin, La (Ni0.5Mn0.5)O3(LNMO-850),LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein.Experimental results showed that the TMP has a higher rate of loaded drug and encapsulation of magnetic nano-particles in the composite, and the modification by Tween80and the encapsulation of magnetic nano-particles showed a significant slow-release properties. Detecting the effect of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay, the results were displayed composite magnetic nano-particles were safe in the appropriate concentration range
In this study, the use of magnetic nano-particles as drug carriers of TMP that provided it for a new dosage form of TCM and has laid a solid foundation, bringing a new dawn for the treatment of migraine.
随着时代的进步,环境的恶化,疾病谱的发展变化,疾病的诊治在现代医学领域里受到了挑战,而中医药在现代临床治疗中逐渐又焕发出新的魅力和迎来新的机遇。中药或中药制剂现已广泛应用于临床,但中药或中药制剂存在着生物利用度低、有效浓度低、起效缓、半衰期短、穿血脑屏障能力差等缺点,严重阻碍其临床疗效的发挥,亦制约了现代中医药的发展。近年来,纳米载药技术已逐渐应用于中医药领域,如中药纳米化、纳米包合技术、聚合物纳米粒载体技术、脂质体纳米粒、固体分散技术等,但仍处于起步阶段,存在许多亟待解决的问题,如纳米中药的药效不确定性以及可能的毒副作用,纳米中药的有效成分和稳定性难以控制等。诸多中药的有效成分为难溶性物质,口服给药吸收差、生物利用度低,故找出合适的方法来提高中药难溶性有效成分的溶解度,改善其生物利用度,是当前中医药工作者面临的主要挑战之一。将中医药复方的有效成分嫁接到磁性纳米微粒上,使其成为生物利用度高、有控缓性能、靶向性的新的药物剂型的研究尚未报道,本课题将从川芎嗪(复方正天丸君药川芎的主要药物成分)的载药开始研究,逐步寻找中医药复方完美的给药途径。磁性纳米材料与中药单体川芎嗪结合组成复合纳米微球,能很好地改善生物利用度低、起效缓、血药浓度低、半衰期短、穿血脑屏障能力差等缺点；磁性纳米粒子具有超顺磁性、小尺寸效应、表面效应、量子隧道效应、磁响应性、生物相容性和生物降解性、功能基团等特性,由于其独特的优势在生命科学领域显示了良好的应用前景。
研究目的：
通过对磁性纳米微粒的制备、表征以及对牛血清蛋白吸附性能测定的综合评价来寻找一种相对有很好载药潜质的并具有超顺磁性、靶向性和生物相容性等优异特性的磁性纳米微粒作为中药的载体,以便更好地改善中药或中药制剂的生物利用度低、起效缓、血药浓度低、半衰期短、穿血脑屏障能力差等缺点。为川芎嗪寻找一种新的剂型,为偏头痛治疗寻找新的良方。
研究方法：
1.磁性纳米粒子铁酸钴镍(Ni0.5Co0.5Fe2O4)的制备、表征及其牛血清蛋白(BSA)吸附性能的测定。
1.1采用共沉淀法制备Ni0.5Co0.5Fe2O4(NCFO)纳米粉体,以NiCl2·6H2O(98%), CoC4H6O4·4H2O(99.5%)和Fe(NO3)3·9H2O(98.5%)为前驱物,将前驱物按所需的化学计量比分别溶于去离子水中搅拌,然后再将三者混合,将混匀的混合溶液加热至800C恒温,搅拌2小时(h)后,一边搅拌一边往溶液中加入氨水,直到溶液的酸碱度(pH)值约等于8.5,停止加入氨水,然后再搅拌大约30分钟后,再加入酒精球磨24h,最后放置干燥箱中干燥后得到所需的前驱体粉体。将得到的粉体样品在通氮气(N2)气氛下并不同温度(5500C到10500C)下退火。
1.2将制备所得的纳米粉体Ni0.5Co0.5Fe2O4样品采用X射线衍射(XRD,(CuKα：λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对合成的样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子Ni0.5Co0.5Fe2O4,然后将其置于铜网上晾干,用透射电子显微镜(TEM,HITACHIH7650日本)观察磁性纳米粒子Ni0.5Co0.5Fe2O4的形态和粒径。
1.3磁性纳米粒子Ni0.5ECo0.5Fe2O4对牛血清蛋白的吸附性能的测定。具体方法如下：将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中配制成pH等于7.4、浓度为1.000mg/ml的溶液,将适量的磁性纳米粒子Ni0.5Co0.5Fe2O4溶于配制好的牛血清蛋白溶液中,将此混合溶液在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米粒子Ni0.5Co0.5Fe2O4对牛血清蛋白的吸附性能。
2.磁性纳米粒子镍锰酸镧(La2NiMnO6)的制备、表征及其牛血清蛋白(BSA)的吸附性能的测定。
2.1采用化学共沉淀法制备La2NiMnO6纳米粉体。具体实验步骤如下：以硝酸镧(La(NO3)3·5H2O(99.5%)),醋酸镍(Ni(CH3COO)2·4H2O(98%))和醋酸锰(Mn(CH3COO)4·4H2O(99%))为前驱物,将前驱物按所需的化学计量比分别溶于去离子水中搅拌,然后再将三者混合,将混合溶液加热至80℃恒温,搅拌2小时后一边搅拌一边往溶液中加入氨水,直到溶液的pH值约等于8.5为止。搅拌大约30分钟后,再加入酒精球磨24小时,然后放置干燥箱中干燥后得到所需的前驱体粉体。将得到的粉体在氮气(N2)气氛下不同温度(750℃,850℃,950℃,1050℃)下退火。
2.2退火获得的磁性纳米粉体La2NiMnO6的表征。退火获得粉体样品的采用X射线衍射(XRD,(CuKα:λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对获得粉体样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子La2NiMnO6然后置于铜网上晾干,用透射电子显微镜(TEM, HITACHIH7650日本产)观察磁性纳米粒子La2NiMnO6的形态和粒径。
2.3磁性纳米粒子La2NiMnO6对牛血清蛋白的吸附性能的测定。具体实验步骤如下：将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中配制成pH等于7.4、浓度为1.000mg/ml的溶液,将适量的磁性纳米粒子La2NiMnO6溶于已配制好的牛血清蛋白溶液中,将此混合溶液在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米微粒La2NiMnO6对牛血清蛋白的吸附性能。
3.磁性纳米粒子铁酸锌镍(Ni0.5Zn0.5Fe2O4)的制备、表征及其牛血清蛋白(BSA)的吸附性能的测定。
3.1采用共沉淀法制备Ni0.5Zn0.5Fe2O4纳米粉体,以醋酸镍(Ni(CH3COO)2·4H2O(98%)),醋酸锌(Zn(CH3COO)2·2H2O(99%)),和硝酸铁(Fe(NO3)3·9H2O(99%))为前驱物,将Ni(CH3COO)2·4H2O(98%)和Zn(CH3COO)2·2H2O(99%)溶于36%的乙酸中组成混合溶液甲,将Fe(NO3)3·9H2O(99%)溶于去离子水组成混合液乙,然后再将甲混合液逐滴加入乙溶液中,边滴边用磁力搅拌,同时加热至80℃后恒温,继续将混合溶液磁力搅拌2小时后,一边搅拌一边往混合溶液中加入氨水,直到溶液的pH值约等于8.5为止。然后静止沉淀、自然冷却后过滤,然后放置干燥箱中干燥24小时,取得干燥混合物再加入酒精球磨后预烧,之后再加入酒精球磨后在通氮气(N2)气氛下并不同温度(400℃,500℃,600℃,700℃,800℃)下退火。
3.2退火获得的磁性纳米粉体Ni0.5Zn0.5Fe2O4的表征。采用X射线衍射(XRD,(CuKα:λ=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02°/2s,衍射角从10°-80°。在30kOe的磁场中,采用振动探针式磁强计VSM(MPMS,美国QUANTUM DESIGN公司)对退火所获得的磁性纳米粉体Ni0.5Zri0.5Fe2O4的样品进行了磁性能的测试。用无水乙醇分散磁性纳米粒子Ni0.5Zn0.5Fe2O4然后将其置于铜网上晾干,用透射电子显微镜(TEM,HITACHIH7650日本)观察磁性纳米粒子Ni0.5Zn0.5Fe2O4的形态和粒径。
3.3采用紫外线分光光度计(UV2401pc)来分析磁性纳米粒子Ni0.5Zn0.5Fe2O4对牛血清蛋白的吸附性能,将牛血清蛋白(BSA,纯度>99%))溶解在去离子水中分别配制成pH等于7.0和3.0浓度为1.000mg/ml的牛血清蛋白溶液,将适量的磁性纳米粒子Ni0.5Zn0.5Fe2O4溶于牛血清蛋白溶液中,将此混合物在常温下超声搅拌2小时,静止沉淀24小时后,用紫外线分光光度计(UV2401pc)来测定磁性纳米粒子Ni0.5Zn0.5Fe3O4对牛血清蛋白在不同酸碱度的环境下(pH=7.0,3.0)的吸附性能。然后选择吸附力强的pH环境的磁性纳米粒子Ni0.5Zn0.5Fe2O4样品,在常温下超声搅拌不同的时间(30-120min),来观察搅拌时间对牛血清蛋白的吸附性能的影响。
4.吐温-80修饰的荧光标记的磁性纳米粒子与川芎嗪组成的复合纳米微粒的制备、表征及其性能的测定。
4.1采用共沉淀法制备荧光粉体LaPO4:Eu,将称量好的Y2O3, La2O3和Eu203溶于l0ml浓硝酸+10m1去离子水混合溶液中,然后加热至80℃C,30分钟后全溶,随后将(NH4)2HPO4直接加入,充分搅拌溶解后,调节PH值为9.5,再继续搅拌2小时。过滤后用去离子水反复冲洗,然后放在干燥箱中烘24小时,研磨,预烧。最后在不同温度下退火。
4.2复合纳米微粒的制备。具体实验步骤如下：用2毫升(m1)浓度为25%的牛血清白蛋白加入20mmg四甲基吡嗪(川芎嗪TMP),再加入30mg磁性纳米粒子镍锰酸镧(La2NiMnO6退火温度为850摄氏度的样品)、15mg荧光粉体掺铕磷酸镧(LaPO4:Eu退火温度为800摄氏度的样品)充分搅拌,再将搅拌均匀的混合物加入40m1的吐温80中,在常温下超声乳化20分钟(min),再取100m1吐温80加热到120摄氏度,将加热的吐温80置于加热器上维持恒温,将超声乳化后的混悬液以100滴／分钟的速度滴入振荡的预热的吐温80中,加热振荡维持恒温10min；然后用冰冷却至25℃,用无水乙醚洗涤3次,每次用无水乙醚50m1,然后3000转／分钟(R/min)离心15min,离心后的复合物待自然蒸发干燥后,4℃C下保存备用。
4.3将制备好的复合纳米微粒采用XRD(CuKα:A=1.54056A)技术来表征,所有样品的XRD的扫描步长为0.02。／2s,衍射角从10。-80。。在30kOe的磁场中,采用VSM(MPMS,美国QUANTUM DESIGN公司)对合成的复合纳米微球样品进行了磁性能的测试。用无水乙醇分散复合纳米粒子,然后置于铜网上,高倍电子显微镜观察复合纳米粒的形态和粒径。
4.4使用荧光光度计测量复合纳米微粒中川芎嗪(TMP)的含量,将复合纳米微粒样品按1:5(mg/ml)加入浓度为5%的稀盐酸溶液中,在超声混匀后8℃下静置24小时,然后2000r/min离心l0min,取上清液,用荧光光度计测量川芎嗪含量。
4.5复合磁性纳米微粒中川芎嗪(TMP)体外释放测定；精密称取制备好的复合纳米微粒,加入适量磷酸盐缓冲溶液(含0.2%叠氮钠作为抑菌剂,0.1%Tween-80作为润湿剂)为释放介质,置于恒温水浴摇床中,在100r/min振荡速度、37℃条件下进行复合纳米微粒中川芎嗪的体外释放速度测定。分别在设定时间(0.5h、1h、2h、5h、10h、15h、20h、25h、30h、35h、40h)取出,于15000r/min离心15min,吸出上清液后,加入等量新鲜的释放介质。采用荧光光度计测量川芎嗪的含量。计算川芎嗪在磷酸盐缓冲溶液(pH7.4,37℃)中的累积释药百分率,以累积释药百分率对时间作图。
4.6复合磁性纳米微粒的细胞毒性测定,采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力影响。
4.7数据分析：实验结果计量资料用均数±标准差(x±s)表示,细胞毒性实验中,实验组与对照组比较采用单因素方差分析,用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义；累积释药百分率散点图采用曲线拟合法拟合。
结果：
1.通过化学共沉淀法成功制备磁性纳米粒子Ni0.5Co0.5Fe2O4(NCFO)。X射线衍射(XRD)分析显示各个不同退火的磁性纳米微粒NCFO样品全都为典型的单相立方尖晶石结构,随着退火温度从550上升到950℃C,磁化强度(Ms)从35.95增加到67.19emu/g。当退火温度进一步升高至1050℃时,Ms有所减小。NCFO纳米粒子的饱和磁化强度和晶粒尺寸最初均随退火温度的上升而增加,后又随着退火温度的上升而有所减小。用透射电子显微镜(TEM, HITACHIH7650)来测定磁性纳米粒子NCFO的粒径,所测得的平均粒径约为50nm。磁性纳米粒子NCFO展现出了对牛血清蛋白有良好的吸附性能,当退火温度处于750℃其粒径33.3nm的磁性纳米微粒NCFO样品显示最强的吸附力为71(mg/g)左右。
2.磁性纳米粒子La2NiMnO6(LNMO)采用化学共沉淀法成功制备。LNMO的晶粒尺寸极大地受到退火温度的影响。随着退火温度从750℃升高到1050℃,它的平均晶粒尺寸从33.9nm增加到了39.6nm,另一方面,矫顽力随着退火温度的升高先增加,950℃退火,平均晶粒尺寸为37.9nm样品的矫顽力达到最大42.30e,然后随着退火温度的继续升高,矫顽力随之减少。该LNMO纳米微粒表现出了对牛血清白蛋白良好的吸附性能,磁性纳米粒子LNMO在850℃温度下退火样品的BSA吸附能力最强,大约为219.6mg/g。在此情况下,吸附后BSA溶液的体积增加了约3m1。
3.采用化学共沉淀法成功制备了磁性纳米粒子Ni0.5Zn0.5Fe2O4(NZFO),通过XRD技术来表征,NZFO粉末形成单一的立方尖晶石结构,并且没有其它杂相的产生。退火温度对磁性纳米粉体Ni0.5Zn0.5Fe2O4晶粒尺寸与磁性能都有一定的影响。随着退火温度的增加,饱和磁化强度增强。在牛血清蛋白溶液的pH=7.0的环境下,退火温度为600℃时的磁性纳米粒子Ni0.5Zn0.5Fe2O4样品对牛血清蛋白的吸附性能为最强,其吸附值约为38.35mg/g。
4.经过吐温-80修饰、掺铕磷酸镧(LaP04:Eu)荧光粉体标记的复合磁性纳米微粒(川芎嗪单体、牛血清白蛋白、La(Ni0.5Mn0.5)O3(LNMO-850)、LaPO4:Eu-800、吐温80共同组成的复合物)采用高温使蛋白质固化的原理成功制备。经高倍电子显微镜观察复合纳米微球的粒径约为0.5um,并且测得其有较高的载药量和包封率。通过对复合磁性纳米微粒的体外释放的测定发现,川芎嗪经吐温80修饰和磁性纳米粒子包封后呈现出明显的缓释性能。数据通过曲线拟合,拟合度为0.56476,结果显示复合磁性纳米微粒的半衰期约为25小时。采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力的影响,经统计分析,纳米药物组与对照组对HepG2人肝癌细胞株增殖活力比较差异无统计学意义(F=3.153P=0.150),结果显示,在一定浓度范围内复合磁性纳米微粒是安全的。
结论：
通过对铁酸钴镍(Ni0.5Co0.5Fe2O4)、镍锰酸镧(La2NiMnO6)、铁酸锌镍(Ni0.5Zn0.5Fe2O4)等三种磁性纳米粒子的制备、表征及其牛血清蛋白吸附性能的测定的综合评价后,选择了在氮气气氛中退火温度为850℃的镍锰酸镧作为制备复合纳米微粒的备选磁性纳米粒子样品。经吐温80修饰、掺铕磷酸镧(LaPO4:Eu)荧光粉体标记的复合磁性纳米微粒(川芎嗪单体、牛血清白蛋白、La2NiMnO6(LNMO-850)、LaP04:Eu-800、吐温80共同组成的复合物)采用高温固化的原理成功制备。实验结果表明川芎嗪在复合磁性纳米微粒中有较高的载药量,且经吐温80修饰、磁性纳米粒子包封后呈现出明显的缓释性能。采用MTT法检测复合磁性纳米微粒对HepG2人肝癌细胞株增殖活力的影响,结果显示在适当的浓度范围复合磁性纳米微粒是安全的。本研究利用磁性纳米粒子为载体成功载药中药单体川芎嗪,为川芎嗪提供了一种新的剂型,为中药复方载药打下了坚实的基础,为偏头痛的治疗带来了新的曙光。
Background:
With the progress of time, environmental degradation and the development of spectrum of disease, diagnosis and treatment of diseases in the field of modern medicine has been challenged. However, the modern clinical medicine in the treatment is gradually fulled of new charm and new opportunities.Nowadays, traditional Chinese medications and its processed drugs are widely used in clinical practice,but they are same shortcomings such as low bioavailability, low effective concentration, slow onset, short half-life, and penetrating poorly blood-brain barrier,which not only seriously hinder drugs effectiveness, but also restrict the development of modern medication.
In recent years, drug-loaded technology of nanoparticles used in the medical field, for example, nano-medicine, nano-inclusion technique, polymer nano-particles carrier technology, liposome nano-particles, solid dispersion technology and other fields, these are still in early stage and many problems need to be solved,including uncertainty efficacy of nano-medicine and possible side effects, difficultly control on active ingredients and instability of nano-medicine.Because of many of poorly soluble active ingredients of traditional Chinese medication, poor oral absorption and low bioavailability,therefore, it is necessary to find the right way to improve the solubility of poorly soluble active ingredients of traditional Chinese medication and its bioavailability, which is one of the main challenges faced by medical workers.
The research that the active ingredients of Chinese herbal compound are grafted onto magnetic nano-particles in order to enhance bioavailability and the performance of controlled slowly drugs and the formulation of targeted new drug has not been reported,This paper will start with one of the major drug with ligustrazine (TMP)of Zheng Tian Pill,gradually finding more perfect route of administration of traditional Chinese medication.Integrating magnetic nano-materials to medicine TMP monomers together form a composite nano-spheres that can well improve defects such as low bioavailability, slow onset, low plasma concentration, short half-life, the poor ability to wear blood-brain barrier.As magnetic nano-particles have super-paramagnetic, small size effect, surface effect, quantum tunneling effect,magnetic response,biocompatibility and biodegradability, functional groups and other characteristics, its unique advantages in the field of life sciences show a promising prospect.
Objective:
Based on the preparation, characterization of magnetic nanoparticles and the comprehensive evaluation for the determination of bovine serum albumin adsorption performance to look for a relatively good magnetic nano-particles with super-paramagnetic, targeting and biocompatibility excellent properties as a carrier of traditional Chinese medicine in order to better improve the low bioavailability of traditional Chinese medication or its processed drugs, slow efficacious, low plasma concentration,the short half-life,badly wearing blood-brain barrier,etc,which can help look for a new dosage of TMP and a new recipe of the treatment of migraine headache.
Methods:
1.The preparation and characterization of nickel Cobalt ferrite(Ni0.5Co0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
1.1The Nio.5Co0.5Fe2O4nano-composites were synthesized by co-precipitation, using NiCl2·6H2O (98%), CoC4H6O4·4H2O(99.5%), and Fe(NO3)3·9H2O (98.5%) as starting raw materials without further purification. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5. After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in N2atmosphere for2h at different temperatures in the range of550～1050℃.
1.2The crystalline phase of Ni0.5Co0.5Fe2O4nano-composites was identified by the x-ray diffraction (XRD) technique. X-ray diffraction gram of all the samples from10°to80°at a scanning step of0.02°/2s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (1=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM) at a room temperature under a maximum field of30kOe.
1.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>98%)1.000mg/ml before the adsorption and for each measurement,3.00to15.00mg nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the aqueous BSA solution for1h at room temperature.
2.The preparation and characterization of Lanthanum-nickel-manganate(La2NiMnO6) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
2.1The La2NiMnO6(LNMO) nano-composites were synthesized by co-precipitation, using La(NO3)3-5H2O(99.5%), Ni(CH3COO)24H2O (98%) and Mn(CH3COO)4-4H2O(99%) as starting raw materials. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h,aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5.After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of750～1050℃.
2.2The crystalline phase of LNMO nano-composites was identified by the X-ray diffraction (XRD)technique. X-ray diffractogram of all the samples from10°to80°at a scanning step of0.02°/s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (X,=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA)at a room temperature under a maximum field of30kOe. The structural defects in LNMO materials were investigated using J EOL4000EX high resolution transmission electron microscope (HR-TEM) operated at400kV.
2.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,La(Ni0.5Mn0.5)O3nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution for1h at room temperature,which put in static precipitation condition after12h to be measured.
3.The preparation and characterization of nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).
3.1The Nio.5Zno.5Fe204nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. The Ni0.5Zn0.5Fe2O4nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. Before both solutions were dissolved in acetic acid (CH3COOH36%), and the latter were dissolved in deionized water, then the former mixed solutions drip into the latter mixed solutions dropwise and stirred meanwhile in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the last mixed solutions, until brown suspension took shape at pH-8.5. The obtained suspension were filtrated and dried in a cabinet dryer at80℃overnight to obtain the precursor samples, which was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough after presintering. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of400-800℃.
3.2Characterizations of Ni0.5Zn0.5Fe2O4nano-particles. The structure and morphologies of the resultant nano-particles were determined by X-ray diffraction (XRD) and transmission electron microscopy (TEM,HITACHIH-7650). The magnetic properties were measured by using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.
3.3The adsorption of bovine serum albumin (BSA (V)) protein on the Nio.5Zno.5Fe204nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.0and3.0contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,6.00to13.00mg of the Ni0.5Zn0.5Fe2O4nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution (pH about7.0and3.0) for1h at room temperature, and which the strongest adsorption was stirred ultrasonically in the different time, which put in Static precipitation condition after12h to be measured.
4. To measurement on the preparation, characterization and properties composited nano-particles combining modified tween80fluorescently was labeled magnetic nano-particles of and TMP
4.1The LaYPO4:Eu powder were synthesized by co-precipitation, using Y2O3, La2O3and Eu2O3as starting raw materials were dissolved in mixed solution of HNO3(69%,10ml) and deionized water(10ml) in required stoichiometric proportions. The mixed solutions were heated to80℃. After2h,(NH4)2HPO4solution was added to the container, until the suspension took shape at pH=9.5. After stirring for about2h the suspension was filtered and then rinsed repeatedly with deionized water. Afterwards dried in a cabinet dryer at80℃overnight to obtain the precursor samples. After grinding and presintering, the dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of500～800℃.
4.220mg Ligustrazine(TMP) add in the bovine serum albumin (BSA(v)25%,2ml).30mg La(Ni0.5Mn0.5)O3(LNMO950℃) nano-particles and15mg LaYPO4:Eu powders join in the mixed solution,then string fully. Compound of after string join in40ml Polysorbate,then the admixture was stirred ultrasonically in the BSA solution for10min at room temperature.100ml heated to120℃to maintain a constant temperature in heater.after the suspensions were ultrasonic stirred dropping in Polysorbate of with maintaining a constant temperature100drop/min speed and vibrate10min.Then was cooled to25degree with ice. With deionized water washing three times, each time with deionized water50ml,centrifuge15min with3000R/min,After centrifugal composite nano-particles to natural evaporation drying,4degrees to save backup.
4.3The structure and morphologies of the resultant nanoparticles were determined by X-ray diffraction (XRD) of all the samples from10°to80°at a scanning step of0.02°/2s was recorded.The magnetic properties were measured by using a vibrating sample magnetometer (VSM, quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.Dispersed composite nano-particles with ethanol, and then placed in a copper-line, high-powered electron microscope composite nano-particle morphology and particle size.
4.4First,use fluorometer to measure the content of TMP in composite nano-particles then,samples of composite nano-particles by1:5(mg/ml) was added at a concentration of5%of dilute hydrochloric acid solution and allowed to stand at8℃after mixing ultrasound for24hours.Last,2000r/min centrifugal10min, the supernatant was used to measure the TMP content by fluorescence photometer.
4.5Composite magnetic nano-particles TMP (TMP) in vitro release assay;prepared composite nano-particles by accurately weighed was added the appropriate amount of phosphate buffer solution (containing0.2%sodium azide as an antimicrobial agent,0.1%Tween-80as a wetting agent) using for the release of the medium,which was placed in a water bath shaker at100r/min shaking speed,37℃condition in order to determinate the vitro release rate of composite nano-particles TMP. Respectively, in the set time (0.5,1,2,5,10,15,20,25,30,35, and40h) removed by centrifugation at15000r/min15min, After aspiration of the supernatant was added an equal amount of fresh release medium. The content of TMP using fluorescence spectrometer measurements. TMP in phosphate buffer solution is calculated(pH7.4,37℃) cumulative release percentage and cumulative release percentage plotted against time.
4.6Cytotoxicity determination of the composite magnetic nano-particles, detecting the influence of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay.
4.7Data analysis:The results of measurement data with mean±standard deviation (x±s) is that the cytotoxicity experiments, the experimental and control groups were compared using ANOVA with SPSS13.0statistical software for analysis, P<0.05set for the difference was statistically significant, using curve fitting fit scatter plot.
Result:
1. The Ni0.5Co0.5Fe2O4(NCFO) nano-particles have been successfully prepared by the chemical co-precipitation process. XRD analysis showed that the each of sample magnetic nano-particles NCFO were all the typical single-phase cubic spinel structure,with the annealing temperature rises from550to950℃, magnetization Ms from35.95to67.19emu/g, when the annealing temperature was further increased to1050℃, Ms decreased. NCFO nano-particles of Grain size and saturation magnetization were initially increased with increasing annealing temperature, and then reduced with increasing annealing temperature and determined the average particle diameter of magnetic nano-particles in NCFO using transmission electron microscope ending with The average particle diameter of about50nm.NCFO magnetic nano-particles showed a bovine serum albumin had good adsorption properties, when the annealing temperature was750℃particle size of magnetic nano-particles NCFO33.3nm revealed the strongest suction force is71(mg/g) or so.
2. La2NiMnO6(LNMO) nano-particles have been successfully prepared by the by the chemical co-precipitation process.The grain size of the LNMO nano-particles are largely influenced by annealing temperature.As the annealing temperature increases from750to1050℃, the average grain size increases from about33.9to39.6nm, On the other hand, the coercivity initially increases, reaching a maximum value of42.3Oe when the average grain size was about37.9nm at950℃, and then reduces. The LNMO nano-particles showed good adsorption performance in bovine serum albumin protein, and the preliminary optimized adsorption was obtained for the LNMO nano-particles annealed at850℃.These LNMO nano-particles were a potential carrier for large biomolecules, which will be widely used in the biomedical field.
3.Successfully prepared by chemical magnetic nano-particles Ni0.5Zn0.5Fe2O4(ZNFO) by co-precipitation method,Characterized by XRD technique, NZFO powder formed a single cubic spinel structure, and do not produce other miscellaneous phase.Annealing temperature on the grain size of magnetic nanoparticles and magnetic powder NZFO had some impacts. With increasing annealing temperature, the saturation magnetization was enhanced. When bovine serum albumin solution at a pH of7.0environment, the annealing temperature was600℃, magnetic nano-particles NZFO samples had the strongest adsorption performance of bovine serum protein, that is, the adsorption value of about38.35mg/g.
4. After modification of Tween80, europium-doped lanthanum phosphate (LaPO4: Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin V,La(Ni0.5Mn0.5)O3(LNMO-850), LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein. The size of composite nano-pheres was about0.5μm and obtain higher drug loading and encapsulation efficiency by high-powered electron microscope. In vitro by measuring the release of the composite magnetic nano-particles and found that TMP modified by Tween80and magnetic nano-particle entrapment showed a significant slow-release properties. Data by curve fitting, the fit is0.56476. The results showed that the half-life of the composite magnetic nano-particles was about25h. Composite magnetic nano-particles to detect the impact of human hepatoma cell line HepG2proliferation activity by MTT assay showed that the composite magnetic nano-particles are safe. After statistical analysis, both nano-drug group and the control group showed no significant difference in HepG2human hepatoma cell line proliferation activity.
Conclusion:
the preparation and characterization of three magnetic nano-particles such as Iron-nickel-cobalt(Ni0.5Co0.5Fe2O4), nickel lanthanum manganite (La2NiMnO6), nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4), and the measurement and evaluation on bovine serum albumin the of adsorption performance. Alternatively magnetic nano-particles selected samples in a nitrogen atmosphere, lanthanum-nickel-manganese annealing temperature of850℃as the preparation of the composite nanoparticles.After modification of Tween80, europium-doped lanthanum phosphate (LaPO4:Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin, La (Ni0.5Mn0.5)O3(LNMO-850),LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein.Experimental results showed that the TMP has a higher rate of loaded drug and encapsulation of magnetic nano-particles in the composite, and the modification by Tween80and the encapsulation of magnetic nano-particles showed a significant slow-release properties. Detecting the effect of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay, the results were displayed composite magnetic nano-particles were safe in the appropriate concentration range
In this study, the use of magnetic nano-particles as drug carriers of TMP that provided it for a new dosage form of TCM and has laid a solid foundation, bringing a new dawn for the treatment of migraine.
引文
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