【作者】 赵淑华；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2009， 博士

【摘要】 磁性纳米粒子不但具有普通纳米粒子所具有的效应（体积效应、表面效应、量子尺度效应和宏观量子隧道效应）,还会随着磁性颗粒材料的组成变化而呈现异常的磁学性质。同时,在许多的生物体内存在以Fe₃O₄为主的强磁性矿物粒晶,所以磁性纳米粒子被认为具有良好的生物相容性,在医学领域的应用有着巨大的优势。但其安全性也受到广泛关注。已有研究表明,磁性纳米粒子因粒径不同、表面包覆情况不同、进入机体的途径不同,显示的毒性不同,从体内排出的时间也不同。目前,超顺磁纳米粒子在临床应用前景主要是载药体系、MRI对比剂等,在这些用途中,纳米粒子进入机体的主要途径为静脉注射,虽然粒子表面包覆后,在较短时间内毒性很低,但其在体内代谢后可能还会在体内存留较长时间。这种状态的粒子对机体产生的作用深受人们关注。本研究以化学共沉淀法制备的Fe₃O₄纳米粒子为材料,对其在不同溶液中的稳定性、静脉注射后的急性毒性和粒子的器官分布以及心肌毒性进行观察,为其安全应用提供基础数据。结果表明,注射用的Fe₃O₄纳米颗粒的平均粒径为9nm,反晶尖石结构。纳米粒子在盐溶液中的稳定性较差,而在高纯水中及含蛋白的溶液中稳定性良好。在急性毒性实验中,小鼠尾静脉1次注射后,LD₅₀为163.60 mg/kg,LD₅₀的95%可信限为147.58 181.37 mg/kg。死亡鼠表现出明显的乏氧体征,可能因呼吸衰竭而死。纳米粒子可分布于全身各器官,主要分布在肺、肝、脾,并可能经肝脏排除。病理学观察表明,纳米粒子对心肌和肾脏产生损害作用,且出现心肌的损害作用较肾脏早。心肌毒性实验中,纳米颗粒1次注射后的不同时间内,小鼠血清LDH、AST的活性下降, TIBC含量增高,心肌细胞的能量代谢酶ATPase活性下降,心肌产生脂质过氧化损伤,心肌细胞发生凋亡,与凋亡密切相关的蛋白表达增加。综上所述,在本实验条件下,未经包覆的Fe₃O₄纳米粒子经静脉注射后可对心肌产生损伤作用。更多还原

【Abstract】 Nano-material as a new kind of material would be applied to a vast number of fields. Nano- biotechnology is highlighted and is a new frontier of the field of international biotechnology, with the specially significant direction of nano- technology advancement in the development of targeted medicine delivery containing nano-material and the implantation of artificial organ created by nano- material. The distinctive way of nano-material contacting human body while applying it in the field of medicine triggers the universal attention and dispute of its safety. This study focus on the stability of Fe₃O₄ nano-particles in different solutions, their acute toxicity to mice and their damage upon cardiac muscle and further to offer basis for the safety application of Fe₃O₄ nano-particles.1. The characterization and stability of Fe₃O₄ nano-particles in different solutionsUtilize transmission electron microscope （TEM） to observe and XRD as well as infrared spectrometer to characterize Fe₃O₄ nano-particles that are synthesized by chemical co-precipitation. The result is that Fe₃O₄ nano-particles obtain cation antisite disorder with an average particle diameter of 9nm.8 kinds of solutions were included, namely, high pure water, 0.1% NaCL, 0.5%NaCL, 0.9% NaCL, RPMI 1640 without blood serum, RPMI 1640 + 5% fetal bovine serum, RPMI 1640 +10% fetal bovine serum, RPMI 1640 + 20% fetal bovine serum, and formulate 5% （V/V） nano-particle solutions combining Fe₃O₄ nano-particles and the ten mentioned solutions. After 3 minutes of exposure to ultrasound, the changes of nano-particle diameter were measured via zeta potential apparatus within five minutes in each of the ten formulated solutions and made the particles in each solution into copper net, and observed the changs of their particle diameter under TEM. The result indicated that in solutions that contained different concentrations of salt, hydrated nano-particles diameter increased rapidly, and the increase rate were relatively unified. However, in the high pure water and fetal bovine serums of different densities, the nano-particles were stable, and no noticeable changes of their particle diameter were observed within five minutes. Moreover, in the solutions containing more than 10% protein, particle’s diameter was close to that with high pure water. The result of TEM observation suggested that despite the fact that hydrated particles’diameter achieved comparatively faster growth in salt solutions, the diameter of those particles themselves showed no changes at all. This was consistent with the situation in the medium with protein. These were all due to the particles aggregation.Based on the discussion above, Fe₃O₄ nano-particles showed good dispersion and stability in high pure water, all of which were mainly associated with their stable electric double layer. Such phenomenon can also be observed in the medium containing blood serum where the reason might be related to the absorbed layer formed by protein on the surface of those particles. Moreover, the extent of dispersion was in accordance with protein density. In those salt solutions the stability of particles was relatively low, which has a possible relationship with the damage of surface electric double layer.2. Acute toxicity of Fe₃O₄ nano-particles in mice and their distribution in its organsUnder bacteria free condition, Fe₃O₄ nano-particle suspension with density of 1.024 mg/ml,1.28 mg/ml,1.60 mg/ml,2.00 mg/ml,2.50 mg/ml respectively were prepared using high pure water. The solutions were subjected to ultrasound for 3 minutes before injecting to mice.120 ICR mice, half male and half female, weighing 18±2g were chosen and divided into six groups, namely, groups with densities of Fe₃O₄ nano-particles of 102.4,128,160,200 and 250mg/kg respectively as well as control group（high pure water）. 20 mouse in each group. Fe₃O₄ nano-particle solutions or high pure water were administered at 0.1 ml/10g via intravenous injection, with a speed of 3⁴sec/0.1ml. The mouse death rate during the next 15 days after the injection were calculated.Extract the survived mouse 20 minutes after the injection as well as 15 days after the injection in 102.4 mg/kg group and 160 mg/kg group, and after anesthetizing, acquire their brains, lungs, hearts, livers, spleens, kidneys and testicles, and fix those organs by 10% formaldehyde. Perform immediate dissection upon those dead mouse in 160 mg/kg group and 250 mg/kg group, and obtain and fix those organs mentioned above, which were then embed using paraffin and cut into slices, followed by dyeing utilizing Prussian blue to observe the distribution in each organ and HE to do pathological inspection on those tissues.The consequence of intravenous injection to test acute toxcity of Fe₃O₄ nano-particles indicated that LD₅₀ of Fe₃O₄ nano-particle was 163.60 mg/kg and the 95% credible line of LD₅₀ was 147.58 ¹81.37 mg/kg. The main behavior of those injected mouse were cramp, rolling, unstable stepping, dyspnoea, and death because of failure in breathing.The result of Prussian coloration indicated that 20 minutes after the injection of nano-particles, the main distribution of those particles was in the liver, spleen, and lung, and considerable reduction of blue particles was seen in the lung and spleen, yet not so in the liver. Low portion of distribution was observed in other organs, say, brain, heart, kidney and testicle. Survived mouse in 160mg/kg group after the injection were seen identical distribution of blue iron particles in the lung, spleen, and liver to that of 102.4 mg/kg group, yet after the dissection of the dead mouse, more dense distribution of the blue particles were discovered on the alveolar wall in the lung, which was also the case in the liver using optical microscope. In contrast to the distribution of blue particles in the liver of the survived mouse, however, blue particles could be observed in the brain, heart and kidney in the dead mouse that were dissected immediately after death. The mouse in 250 mg/kg group immediately perished after the injection. Nano-particles in the lung appeared aggregated distribution, blue substances in the liver appeared the distribution in an even flocculating form, the quantity of particles in the spleen showed apparent reduction and that in the brain were seen obvious growth. Moreover, blue substances were more readily seen in the brain and liver.The result of pathological observation suggested that 20 minutes after the injection, the particles in the cardiac muscle of the survived mouse were seen denaturalized, moreover, the scope of the denaturalized region expanded as the dose of the particles growed. 15 days later, local necrosis could be observed in the cardiac muscle, while such pathological changes did not exist in the dead mouse. Kidney of the mouse in 102.4 mg/kg group 20 minutes after the injection was not seen abnormal; however, brown particle-shaped substance was seen in the capillaries of glomerulus of the survived mouse of 160mg/kg group, yet not so in the rest mouse. 2 weeks later, protein cast could be observed in the glomerulus of the survived mouse of the two groups as well as edema in the carunculae papillaris; however, in the injected dead mouse, blood congestion in the carunculae papillaris and particle shaped substance in the blood vessels in the some of the glomerulutilizes could be seen.Based on the presentation above, after the intravenous injection of 9 nm Fe₃O₄ nanoparticles, LD₅₀ of mouse was 163.60 mg/kg, with 95% credible line of LD₅₀ at 147.58 181.37 mg/kg. The main behavior before the death of mouse was typical breathing difficulty, and Prussian special coloration indicated full covering of blue iron particles on the base of the capillaries of the pulmonary of the dead mouse. On the contrary, the pathological observation （HE coloration） of the lung showed no sign of pathological changes, say, inflammation or bleeding, yet cardiac muscle that was sensitive to oxygen shortage appeared particle denaturalizing, and there was protein cast in the kidney; nano-particles, which was mainly excreted by liver and slow in excretion, performed a rapid distribution in the spleen and liver within a relatively short time （20 minutes）, and the more the dose of the nano-particles, the wider of the scope of their distribution.3. The impact of Fe₃O₄ nano-particles upon the blood serum enzymes and the cardiac musclesPrepare suspension of Fe₃O₄ nano-particles with high pure water under the condition of bacteria free into three groups with the density of 0.1mg/ml,0.2mg/ml,and 0.8mg/ml respectively. Expose those suspensions to ultrasound for 3 minutes.Choose 72 male ICR mouse with weight of 18±2g, then equally divide them into 4 groups, namely, group with Fe₃O₄ nano-particles 10 mg/kg, 20 mg/kg, 80 mg/kg and a control group （high pure water）, and perform intravenous injection of the suspensions of nano-particles at 0.1ml/10g with the speed of 3 4 sec/0.1ml. After 1d, 3d and 7d respectively, randomly choose 6 mouse in each group, fetch blood from the eyes, and extract the blood serum, which would be utilized to measure LDH, AST activity, content of TIBC; fetch heart, liver, spleen to measure their weight and the heart was to be utilized to measure the content of water, oxidization damage, and the activity of enzymes in the cardiac muscle.Divide equally another 20 mouse into 4 groups, and after 1d of injecting them with particles of the densities mentioned above anesthetize them by injecting 10% chloral hydrate of 0.03 mL/10g through abdominal cavity, followed by acquiring the whole heart, then fix it with 10% formaldehyde for 24 h, embedded by using paraffin and slice them into pieces, which were then utilized to examine the apoptosis of cardiac muscle and the related activities of protein and gene expressions.The result was that apparent reduction of the activity of the blood serum LDH and AST occurred 1 day after the injection of nano-particles, moreover, the more the dose of the particles, the more the activity of the enzymes would reduce and no sign of recovery could be seen after 7 d; TIBC in the blood serum rose and groups of low dose were seen earlier rising while that of high dose later, after 7d, all test group were considerably higher than the control group.The content of water in the cardiac muscle decreased tremendously.The content of MDA in the cardiac muscle serum 1d after the injection plummeted, while 3d later that of each experimental group grew remarkably, and that of each experimental group was apparently higher than that of the control group till 7d later. The activity of SOD reduced vastly 1d after the injection, and that of every experimental group was continuously greater than that of the control group on the 3rd day after the injection, and such was also the case till the 7th day. The change of the activity of GSH-Px was similar with that of SOD, yet that of former was later than that of the latter.1d after the injection, the activities of Na⁺, K⁺- ATPase and Ca²⁺, Mg⁺- ATPase in each particle exposure group were considerably higher than that of the control group; 3d later, the activities decreasd obviously; 7 days later, 80mg/kg group was apparently lower than other groups, yet the activity of Na⁺, K⁺-ATPase in 10 mg/kg group and 20mg/kg group recovered to the level of the control group, and the activity of Ca²⁺, Mg²⁺- ATPase in 20 mg/kg group remained lower than that of the control group.1d after the injection, apoptosis of the myocardium cell was determined by means of TUNEL. The results demonstrated that apoptosis of myocardium cell occurred in the injected groups, and the more the amount of particle injected, the more severe the apoptosis emerged. The result of grayscale detection suggested that apparent dose dependence existed, in the meantime, the activities of Caspase-9 and Caspase-3 rose tremendously, and the expressions of Fas、FasL、p53 were much higher than that of the control group.Based on the presentation above, after performing intravenous injection of Fe₃O₄ nano-particles on the mouse, it was likely that the releasing of the inflammation factors, which was caused by breathing difficulty and the nano-particles, resulted in the apoptosis of the cardiac muscle, the behavior of which is that the activity of LDH, AST enzymes that were intimately associated with the metabolism of the cardiac muscle decreased enormously, and the activity of Na⁺, K⁺- ATPase and Ca²⁺, Mg⁺- ATPase on the cell membrane reduced greatly. The two reductions most likely gave rise to the increase of the density of Ca²⁺ in the cells. The amount of expressions of p53、Fas、FasL rose vastly, and rendered cardiac muscle apoptosis by activating Caspase through activating Caspase system.更多还原