2种超顺磁性Fe3O4纳米粒子的制备及其对大鼠肺部的急性毒性效应

Preparation of two kinds of superparamagnetic iron oxide nanoparticles and their acute toxicity to rat lungs

目的:分别制备壳聚糖和油酸钠修饰的超顺磁性Fe3O4纳米粒子(superparamagnetic iron oxide nanoparticles,SPIONs),并探讨其对大鼠肺部的急性毒性效应。方法:采用化学共沉淀法制备壳聚糖和油酸钠修饰的SPIONs并对其进行表征。将54只SD大鼠随机分为生理盐水对照组(n=18)、壳聚糖SPIONs组(n=18)和油酸钠SPIONs组(n=18)。口腔内气管插管给药,对大鼠的一般情况进行观察,分别于给药后24、72 h和第14天随机处死每组中6只大鼠,腹腔静脉取血检测主要血生化指标,取支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),计数细胞总数和中性粒细胞的数量,HE染色观察肺部的病理改变。结果:(1)制备的SPIONs均为30 nm左右的类球形结晶。(2)大鼠一般情况好,未发生死亡。(3)2实验组血清中的主要血生化指标仅有24 h总蛋白(total protein,TP)含量和14 d碱性磷酸酶(alkaline phosphatase,ALP)含量2项指标与正常对照组有差异[TP(g/L)24 h组:壳聚糖SPIONs组(n=6):62.40±3.60,油酸钠SPIONs组(n=6):69.95±5.16,正常对照组(n=6):63.22±2.55,F=6.344,P=0.010;ALP(u/L)14 d组:壳聚糖SPIONs组(n=6):78.83±7.92,油酸钠SPIONs组(n=6):106.83±26.95,正常对照组(n=6):99.85±9.63,F=4.336,P=0.033)。(4)实验组BALF中的细胞总数(×104个)在24 h和72 h均高于正常对照组(壳聚糖SPIONs24 h组(n=6):780.00±130.58,油酸钠SPIONs 24 h组(n=6):1 000.00±166.85,正常对照24 h组(n=6):498.33±114.75,F=19.605,P=0.000;壳聚糖SPIONs 72 h组(n=6):809.17±197.49,油酸钠SPIONs 72 h组(n=6):920.00±125.54,正常对照72 h组(n=6):580.00±123.29,F=7.735,P=0.005),且油酸钠SPIONs组明显高于壳聚糖SPIONs组,在14 d时均下降(壳聚糖SPIONs 14 d组(n=6):628.33±133.29,油酸钠SPIONs 14 d组(n=6):654.17±99.27,正常对照14 d组(n=6):570.83±94.15,F=0.898,P=0.428)。(5)实验组BALF中中性粒细胞的数量(×104个)在24 h和72 h均高于正常对照组(壳聚糖SPIONs 24 h组(n=6):45.00±14.12,油酸钠SPIONs 24 h组(n=6):90.00±24.50,正常对照24 h组(n=6):1.02±0.33,F=44.565,P=0.000;壳聚糖SPIONs 72 h组(n=6):9.15±8.82,油酸钠SPIONs 72 h组(n=6):19.17±5.38,正常对照72 h组(n=6):1.09±0.57,F=13.791,P=0.000),且油酸钠SPIONs组明显高于壳聚糖SPIONs组,在14 d时下降至接近正常。(6)实验组的肺部HE染色可以明显看到肺泡断裂,肺泡壁增厚,炎症细胞浸润,油酸钠SPIONs组炎症表现更严重。壳聚糖SPIONs组肺损伤有自我修复的趋势,而油酸钠SPIONs组减轻不明显。结论:壳聚糖SPIONs与油酸钠SPIONs相比,对肺部毒性作用小,并且随着时间的延长,毒性效应逐渐减轻。

Objective：To prepare superparamagnetic iron oxide nanoparticles（SPIONs）modified by chitosan and sodium oleate and to investigate their acute lung toxicity. Methods：The SPIONs were prepared by coprecipitation and their properties were measured.Fifty-four SD rats were divided into three groups randomly：control group（n=18）,chitosan-SPIONs group（n=18）and sodium oleate-SPIONs group（n =18）. The rats were exposed to SPIONs by intratracheal instillation. General condition of the rats was observed. Six rats of each group were killed randomly at 24 h,72 h and 14 d after the exposure. The major blood biochemical indicators were analyzed with an automatic biochemical analyzer. Bronchoalveolar lavage fluid（BALF）was taken and the total cells and neutrophil cells were counted. The pathological changes of lungs were observed with HE staining. Results：1Both of the SPIONs exhibited structure of spherical crystallization with a diameter of30 nm. 2The normal situation of rats was good and no rat died;3Only two blood biochemical indicators including total protein（TP）of 24 h and alkaline phosphatase（ALP）of 14 d were different from those of control group（TP（g/L）24 h group：chitosan-SPIONs group（n=6）：62.40±3.60,sodium oleate-SPIONs group（n=6）：69.95±5.16,control group（n=6）：63.22±2.55,F=6.344,P=0.010;ALP（u/L）14 d group：chitosan-SPIONs group（n=6）：78.83±7.92,sodium oleate-SPIONs group（n=6）：106.83±26.95,control group（n=6）：99.85±9.63,F=4.336,P=0.033）. 4Total cells（×10^4）in BALF of experimental groups were higher than those in control group at 24 h and 72 h and total cells of sodium oleate-SPIONs group were obviously higher than those of chitosan-SPIONs group（chitosan-SPIONs 24 h group（n=6）：780.00±130.58,sodium oleate-SPIONs 24 h group（n=6）：1 000.00±166.85,control 24 h group（n=6）：498.33±114.75,F=19.605,P=0.000;chitosan-SPIONs 72 h group（n=6）：809.17±197.49,sodium oleate-SPIONs 72 h group（n=6）：920.00±125.54,control 72 h group（n=6）：580.00±123.29,F=7.735,P=0.005）. Both of them were decreased to normal on 14 d（chitosan-SPIONs group（n=6）：628.33±133.29,sodium oleate-SPIONs group（n=6）：654.17±99.27,control group（n=6）：570.83 ±94.15,F=0.898,P =0.428）. 5Neutrophil cells（×10^4）in BALF of experimental groups were higher than those of control group at 24 h and 72 h（chitosan-SPIONs 24 h group（n=6）：45.00±14.12,sodium oleate-SPIONs 24 h group（n=6）：90.00±24.50,control 24 h group（n=6）：1.02±0.33,F=44.565,P=0.000;chitosan-SPIONs 72 h group（n=6）：9.15±8.82,sodium oleate-SPIONs 72 h group（n=6）：19.17±5.38,control 72 h group（n=6）：1.09±0.57,F=13.791,P=0.000）. Neutrophil cells in BALF were more in sodium oleate-SPIONs group than in chitosan-SPIONs group. 6HE staining showed the fracture of pulmonary alveoli,thickening of alveolar walls,and inflammatory cell infiltration in the experimental groups which were more serious in sodium oleate-SPIONs group than in chitosan-SPIONs group. Conclusion：Chitosan modified SPIONs have less toxicity to the rats＇ lungs than sodium oleate modified SPIONs and their lung toxicity reduces over time.