CN114534692A - Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用 - Google Patents
Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
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Abstract
本发明公开了一种Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用,本发明Fe3O4@SiO2@DMSA磁性纳米材料细胞毒性低,血液相容性好,无明显的免疫抑制或增强,无明显的脏器毒性,吸附后理化性质稳定、体外Pb吸附率高等均表明其作为临床血液灌流吸附剂的有效性及可行性。
Description
技术领域
本发明涉及一种Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除重金属铅离子中的应用,还涉及该Fe3O4@SiO2@DMSA磁性纳米材料作为灌流吸附剂的应用。
背景技术
铅是在我国广泛应用的工业原料且不可降解,铅污染长期存在于生产和生活环境中,进入人体后在组织器官中蓄积并产生毒性,引起贫血、肾损伤、心血管损害和智力行为异常等,严重威胁人类的健康。
目前Pb中毒药物治疗以络合剂(如EDTA、DMSA)为主,但肾毒性及通过肾脏排泄局限了其使用。吸附剂用于驱铅研究已取得很大进展,吸附剂不仅可降低机体铅含量,还具有较络合剂更显著的优点,既不增加铅在肾脏排泄也不加重肾脏的铅负荷。
血液灌流是临床上治疗急性重金属中毒最常用的方法之一,血液灌流是将患者的血液引出体外,利用吸附剂吸附去除血液中的重金属等有害物质,使患者的血液得到净化。血液灌流的关键是吸附剂,目前采用的吸附剂主要依靠进口,成本高昂,极大地加重了患者的负担。因此,研究一种制作简单、成本低廉、吸附性能好的血液灌流吸附剂十分必要。
发明内容
针对现有技术存在的不足,本发明提供了一种Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用,该Fe3O4@SiO2@DMSA磁性纳米材料制备简单,成本低廉,具有低的低保毒性和低的溶血液率,对重金属Pb具有很好的吸附率,是一种很有潜力的血液灌流吸附剂。
本发明提供了Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用。
进一步的,本发明还提供了Fe3O4@SiO2@DMSA磁性纳米材料在制备血液灌流吸附剂中的应用,其可以用于吸附去除血液中的铅,治疗急性铅中毒。
进一步的,所述Fe3O4@SiO2@DMSA磁性纳米材料是由2,3-二巯基丁二酸(DMSA)对核壳结构的Fe3O4@SiO2进行改性而得。其中,二氧化硅可以通过现有技术中公开的方式将二氧化硅包覆到Fe3O4表面。
进一步的,所述Fe3O4@SiO2@DMSA磁性纳米材料比表面积大、表面活性中心多、吸附能力高,不仅耦合了Fe3O4@SiO2和DMSA的双重特性,其良好的生物相容性及低细胞毒性也为后期医学应用提供了广阔前景,是一种高效的吸附剂。
本发明具有以下优势:
1、本发明Fe3O4@SiO2@DMSA磁性纳米材料体外血铅吸附研究表明,当铅离子含量从200ppb到1600ppb,Fe3O4@SiO2@DMSA磁性纳米材料吸附剂浓度1-5mg/ml时,该磁性纳米材料吸附率均>50%,能满足临床常见的中重度铅中毒(400 ppb)的治疗。该磁性纳米材料的吸附率在血铅浓度<800ppb时出现最高值,此后平稳,表明该材料对血液中其他成分也可能存在吸附作用。该磁性纳米材料在猪体内灌流吸附率最高为41.55%,低于体外实验,可能是由于灌流器内材料与血液接触不充分、接触面积减小所致。
2、本发明Fe3O4@SiO2@DMSA磁性纳米材料结构经XRD验证与血液接触前后无本质变化,吸附后FTIR显示COOH、OH峰的位移, XRP检测出Pb元素,表明该纳米材料通过羟基、羧基等与血液中Pb发生化学吸附。铅在血液中几乎全部与红细胞内的HGB结合且红细胞内的铅与血浆内铅保持着动态平衡。通过血液循环,铅进入其它组织,因而与组织也保持着动态平衡。血铅降低促使细胞内和组织中的铅向细胞外转移,最终达到新的平衡。慢性中毒95%铅在骨组织中沉积,而骨铅仅一部分与软组织铅、血铅相互转换,因此Fe3O4@SiO2@DMSA更适合急性或中重度铅中毒的治疗。
3、本发明Fe3O4@SiO2@DMSA磁性纳米材料对吸附后外周血红细胞进行SEM检测并未发现材料残留且吸附前后外周血和红细胞中铁元素含量无差异,表明其吸附主要发生在红细胞外。吸附前93.17%的铅位于红细胞,吸附后虽然红细胞内铅浓度降低,但其与血浆铅浓度比例未发生明显变化,表明吸附符合铅动力学分布,且主要发生在红细胞外。
4、本发明Fe3O4@SiO2@DMSA磁性纳米材料细胞毒性低,血液相容性好,无明显的免疫抑制或增强,无明显的脏器毒性,吸附后理化性质稳定、体外Pb吸附率高等均表明其作为临床血液灌流吸附剂的有效性及可行性。
附图说明
图1为Fe3O4@SiO2@DMSA一般性能图,图1A、1B为吸附前后TEM图,图1C为吸附前后XRD图,图1D为材料在吸附前后FTIR变化,图1E为吸附前XPS,图1F为吸附后XPS。
图2体内外铅吸附图。
图3灌注前后对细胞免疫和炎症的影响。(A,C,E)显示CD3+CD45+,CD3+CD4+,CD3+CD8+在血液灌流前的细胞抗原表达水平,而(B,D,F)显示血液灌流后的水平。(G,H,I)表明炎症因子IL-6,TNF-α,CRP在吸附前和吸附后的水平。
图4为血液灌流前后红细胞的扫描电镜图像。血液灌流前红细胞形态,×2.5k(A);与红细胞接触的纳米材料,×4.0 k(B);吸附过程中的红细胞分散度,×2.5 k(C);血液灌流后,通过磁分离分离出纳米材料,×2.0 k(D)。
具体实施方式
下面通过具体实施例对本发明进行进一步解释和说明,下述说明仅是示例性的,并不对其内容进行限制。
实施例
合成Fe3O4@SiO2纳米复合材料:将100 mg制备的亲水性Fe3O4纳米颗粒在乙醇(150 ml)中超声悬浮40 min。依次向悬浮液中添加氨水(3 ml)和去离子水(47 ml),并将混合物(乙醇-水-氨水溶液,75.0/23.5/1.5 v/v/v%)超声处理40分钟。然后,在室温下,在温和连续搅拌下缓慢添加0.6 ml四乙氧基硅烷,再持续8小时。通过磁分离分离分离二氧化硅包覆的纳米复合物,并用乙醇洗涤几次。收集最终产物,并在60°C下真空干燥。
巯基改性Fe3O4@SiO2@DMSA:100毫克Fe3O4@SiO2将纳米颗粒在10毫升甲苯中超声分散30分钟,然后向溶液中添加10毫升二甲基亚砜和250毫克二甲基亚砜。将混合物搅拌12h。通过磁选收集最终产物,用乙醇和去离子水洗涤几次,并在60°C下真空干燥。
对所得磁性纳米材料Fe3O4@SiO2@DMSA的铅吸附性能进行研究,具体如下:
1、材料
1.1实验动物
近交系五指山小型猪12头,雌雄不限,体重约25kg,清洁级(海南省农科院提供,2012BAI39B04)。实验获得海口市动物实验伦理委员会批准,操作符合《实验动物管理条例》。
1.2 实验试剂及设备
戊巴比妥钠(Merck-57330);猪IL-6(E06786p)、猪TNF-β(E16980p)、猪hsCRP(E08163p) ELISA Kit(武汉华美);乙酸铅(Aladdin-L112922)、戊二醛(Aladdin-G105907);四氧化锇(Sigma-201030);MDA试剂盒(A003-1)、SOD试剂盒(A001-3)购自南京建成;CD3(ab16669)、CD4(ab25804)、CD19(ab24936)等购自Abcom公司;CD8(559584)、CD16(551395)、CD56(564489)购自BD公司。日本JEM-2100透射电子显微镜;日本S3000N扫描电镜;日本SysmexCA-1500全自动凝血分析仪;美国Bio-rad X-mark酶标仪;美国BDFACSCanto 流式细胞仪;德国B.Braun血透仪;血液、组织中铅元素、铁元素由广州金域中心检测。
2、实验方法
2.1Fe3O4@SiO2@DMSA性能检测
透射电子显微镜(TEM)观察纳米材料的粒径及形貌;X射线衍射(XRD)观察材料的成分、性质;傅里叶红外(FTIR)观察纳米材料的分子结构;X射线光电子能谱分析(XPS)对材料元素进行定性分析。
分离血铅吸附后纳米材料,0.9%Nacl洗涤后3000rmp/min离心3min,重复操作2次,材料性能检测方法同上。
2.2五指山小型猪铅中毒模型建立
按照5mg/kg 乙酸铅剂量食物染毒。持续2w建立急性中度铅中毒模型(400ppb)。监测静脉血铅水平,成功建模后分别获取猪骨、肝、肾、心、脑、肺等器官组织;所有组织、血液中铅由广州金域中心检测。
2.3体外铅吸附研究
单样本抽取肝素钠抗凝静脉血2ml,将已分散的纳米材料与血液在37℃下接触60min后用磁分离架分离出材料,测血铅浓度。铅吸附率计算如下:
其中,C1初始铅浓度:g/L; C2吸附后铅浓度:g/L。
2.4体内铅吸附研究
制作用于五指山猪的体外灌流器,将纳米材料置于灌流器内,建立体外灌流循环通路。设置血流量 50ml/min;肝素钠持续抗凝;PBE:300mmHg;PV:280mmHg;PFD15mmHg。采集灌流前、灌流60min;术后72h静脉血液,检测血生化、免疫炎症等系统变化。
2.4.1 外周血炎症因子IL-6、TNF-α及CRP检测步骤见S Wu等。细胞表面抗原CD3/4/8/16/19/56等检测步骤参考试剂盒。
2.4.2红细胞SOD检测步骤见Shao-Hua Liu等。红细胞MDA步骤见Yan Deng等。
2.4.3红细胞SEM
取吸附前后外周血各0.5ml,3000rmp/min离心,取沉淀红细胞。2.5%戊二醛固定,1%饿酸后固定;30%、50%、70%、90%酒精梯度脱水2次;乙酸异戊酯置换;CO2临界干燥、喷金后SEM观察红细胞状态。
3、实验结果
3.1Fe3O4@SiO2@DMSA吸附前后性能
TEM显示Fe3O4@SiO2@DMSA粒径约为400nm,吸附前分布均匀(图1A),吸附后团聚稍增多(图1B)。XRD显示Fe3O4@SiO2@DMSA为立方逆尖晶石结构(图1C),吸附后XRD无明显变化(图1C)。FTIR中1619、1360附近波峰归因于COO-基团的非对称和对称拉伸(图1D),吸附后出现羟基峰、羧基峰的位移。XPS检测到Fe、Si、O、S(图1E),吸附后检测到Pb附着(图1F)。
3.2五指山小型猪铅中毒模型的建立
染毒2w后,血铅为(379.5±18.6)ppb。器官中铅含量骨骼中最高;外周血中93.17%铅分布于红细胞,血浆中约为6.83%。吸附后铅分布未发生明显变化,仍有89.56%的铅位于红细胞。建模过程中HGB增高; ALB、GLOB、AST等升高、A/G倒置表明肝功能受损;LDL、APOA、APOB等血脂指标异常可能为建模过程中饮食控制引起; MB增高提示心功能受损;肾功能、电解质无明显变化。
3.3 Fe3O4@SiO2@DMSA体内外铅中毒吸附结果
选择1 mg/ml的浓度进行吸附研究。猪对铅的吸附结果表明,猪在体外的吸附率为55.7±7.3%(图2,A),而猪血液灌流期间的吸附率明显降低至37.8±6.5%(图2,C)。在树脂组中,吸附率在体外为23.4±2.6%,在体内为22.3±1.7%(图2、B和D)。
当我们将处理时间加倍到120分钟时,吸附率增加到41.3±4.6%(图2,E);然而,60分钟和120分钟之间没有显著差异(P=0.23)。此外,随着透析频率的增加,最终铅清除率为71.2±7.5%,血铅水平降至82.7±5.3 ppb。
3.4猪血液灌流期间的体内毒性
模型的血常规结果显示血红蛋白(HGB)水平升高;肝功能结果显示白蛋白(ALB)、球蛋白(GLOB)和天冬氨酸转氨酶(AST)水平升高,ALB/GLOB(A/G)比值倒置,血脂低密度脂蛋白(LDL)、载脂蛋白A和B(APOA和APOB)水平异常。心功能结果显示CK水平升高;这些异常可能是铅中毒引起的。然而,肾功能、电解质和血气分析显示无明显变化。
血液灌流期间生命体征稳定:呼吸频率、心率和体温分别为11-19次/分钟、70-87次/分钟和38.3±0.3°C。实验组术后72小时血常规检测结果显示白细胞(WBC)、血红蛋白(HGB)、红细胞压积(HCT)和血小板(PLT)水平下降,但无差异;然而,在对照组中,只有白细胞水平下降。肝脏没有明显变化,肾、心功能及钠、钙、镁、钾等吸附前后无明显变化; Cl-紊乱可能与灌流过程中补液有关。红细胞MDA、SOD在灌流前后无明显变化。
在外周血淋巴细胞免疫方面,CD3+CD4+/CD3+CD8+细胞的比例在1.57到2.16之间,在正常范围内,血液灌流后没有显著变化(P=0.32)(图3、A-F)。炎症因子IL-6在血液灌流过程中和72小时后均无明显变化,而TNF-α和CRP水平在60分钟血液灌流过程中均升高;血液灌流72小时后,这些指标恢复到正常水平(图3,G-I)。血液灌流分离纳米颗粒后,在红细胞中未观察到残余物质(图4,A-D)。
4、结论
从上述实验结果可以看出,本发明Fe3O4@SiO2@DMSA细胞毒性低、血液相容性好、无明显的免疫抑制或增强、无明显脏器毒性、吸附后理化性质稳定、体外Pb吸附率高等均表明其作为临床血液灌流吸附剂的有效性及可行性。
Claims (4)
1.Fe3O4@SiO2@DMSA磁性纳米材料在吸附去除铅离子中的应用。
2.根据权利要求1所述的应用,其特征是:所述Fe3O4@SiO2@DMSA磁性纳米材料是由2,3-二巯基丁二酸对核壳结构的Fe3O4@SiO2进行改性而得。
3.Fe3O4@SiO2@DMSA磁性纳米材料在制备血液灌流吸附剂中的应用。
4.根据权利要求3所述的应用,其特征是:所述Fe3O4@SiO2@DMSA磁性纳米材料是由2,3-二巯基丁二酸对核壳结构的Fe3O4@SiO2进行改性而得。
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