CN116395747B - 一种四氧化三锰-硫化银Janus结构纳米复合材料及其制备方法 - Google Patents
一种四氧化三锰-硫化银Janus结构纳米复合材料及其制备方法 Download PDFInfo
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- -1 Manganese tetraoxide-silver sulfide Chemical compound 0.000 title claims abstract description 27
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000002105 nanoparticle Substances 0.000 claims abstract description 59
- 239000011572 manganese Substances 0.000 claims abstract description 36
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000003756 stirring Methods 0.000 claims abstract description 26
- QGLWBTPVKHMVHM-KTKRTIGZSA-N (z)-octadec-9-en-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCN QGLWBTPVKHMVHM-KTKRTIGZSA-N 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 20
- 229910052709 silver Inorganic materials 0.000 claims abstract description 14
- 239000004332 silver Substances 0.000 claims abstract description 14
- YUKQRDCYNOVPGJ-UHFFFAOYSA-N thioacetamide Chemical compound CC(N)=S YUKQRDCYNOVPGJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- DLFVBJFMPXGRIB-UHFFFAOYSA-N thioacetamide Natural products CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 12
- HYZQBNDRDQEWAN-LNTINUHCSA-N (z)-4-hydroxypent-3-en-2-one;manganese(3+) Chemical compound [Mn+3].C\C(O)=C\C(C)=O.C\C(O)=C\C(C)=O.C\C(O)=C\C(C)=O HYZQBNDRDQEWAN-LNTINUHCSA-N 0.000 claims abstract description 10
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- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 claims description 15
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- PLKATZNSTYDYJW-UHFFFAOYSA-N azane silver Chemical compound N.[Ag] PLKATZNSTYDYJW-UHFFFAOYSA-N 0.000 claims description 6
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- PPNAOCWZXJOHFK-UHFFFAOYSA-N manganese(2+);oxygen(2-) Chemical compound [O-2].[Mn+2] PPNAOCWZXJOHFK-UHFFFAOYSA-N 0.000 claims description 5
- VASIZKWUTCETSD-UHFFFAOYSA-N manganese(II) oxide Inorganic materials [Mn]=O VASIZKWUTCETSD-UHFFFAOYSA-N 0.000 claims description 5
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Abstract
本发明公开了一种四氧化三锰‑硫化银Janus结构(Mn3O4‑Ag2S)纳米复合材料及其制备方法。属于材料制备技术领域。本发明方法所制备的复合材料形状均一,平均粒径为5‑15nm,具有良好的分散性,生物相容性好。制备方法如下:将乙酰丙酮锰溶于油胺中,在惰性气氛的保护下搅拌加热,离心得到的四氧化三锰纳米颗粒转移至含有曲拉通和油胺的混合溶液中,同时加入银胺溶液,充分搅拌之后加入硫代乙酰胺溶液,混合溶液加热搅拌。反应结束后冷却,用无水乙醇洗涤,离心,分散在环己烷中。最后用F‑127进行表面改性,使该纳米复合材料具有良好的生物相容性。该方法制备的纳米复合材料具有在癌症治疗应用中的核磁共振增强效果和作为光敏剂的潜力。
Description
技术领域
本发明涉及一种纳米复合材料的制备方法,具体涉及一种四氧化三锰-硫化银Janus结构纳米复合材料的制备方法,该方法简单稳定,可广泛应用于生物、医学领域。
背景技术
随着日益严重的人口老龄化问题和逐渐加深的社会压力和环境负担,对于癌症的诊断和治疗从根本上来说依然面临着严峻的挑战。癌症由数百种不同的分子疾病组成,这要求大部分患者在癌症治疗过程中需要具有定制化的调整策略。而个性化的方法针对于特定的癌细胞,需要依赖于适当的分子种类的结合,即所谓的分子靶向治疗。与单畴粒子结构相比,Janus纳米颗粒因为其不对称结构和官能化,一般表现出两种相反但不相互影响的独特特性,为Janus粒子提供了一个从材料科学到生物学和催化的多功能应用平台,同时各种纳米材料组合的构建使Janus纳米颗粒在工程、稳定器、自拉式马达、传感器和药物输送等领域具有优异的应用前景。
近年来,各类学科的有机结合为癌症的诊断和治疗提供了各式各样的研究思路,用以促进对目标人群在癌症疾病不同阶段所需的有效把控,而实现癌症早期阶段的准确诊断成为疾病治疗过程中首要的一环。
其中,癌症的分子成像要求高度敏感的条件,因为在肿瘤组织中异常表达的生物分子的浓度通常非常低(在皮摩尔到纳摩尔范围内)。纳米颗粒是满足这一要求的理想试剂,这是因为纳米材料将肿瘤治疗和成像双重功能集成到一个单一***,有机统一学科之间的优势,实现了从精准数据检测到完整的病变区域成像再到个性化靶向治疗的整个过程。其中Janus纳米颗粒由于相对较小的表面自由能,不仅集合传统复合纳米颗粒的多重性质,同时保留因为空间结构限制的内在性质,还因为复合颗粒界面的独特性质在生物医药领域受到广泛关注。但是其制备手段受限于精密的设备,复杂的操作和苛刻的反应条件,无法实现大规模生产,这为其在临床应用的推广设置了阻碍。
本课题利用Ag2S-Mn3O4 Janus结构纳米颗粒作为目标材料,提出一种简单、经济、绿色的合成手段,制备尺寸和形貌均一的异质体结构。结合核磁共振成像和光热治疗两种手段在癌症病变区域实现药物释放,最终达到协同良好的癌症诊断和治疗的效果。***分析材料形貌和表面电荷等材料学特性,判断生物相容性,光热转换效率以及药物释放能力。结合体外细胞实验和体内动物实验,探究Ag2S-Mn3O4异质体结构作为造影剂的增强效果以及用于癌症材料治疗的安全性和有效性,寻找进一步提高诊断和治疗效果的途径,最终探索纳米颗粒介导的癌症治疗相关机理。
发明内容
本发明的目的在于克服现有技术的不足,提供一种具有核磁共振增强效果和增强光热和光动力疗效的四氧化三锰-硫化银Janus结构纳米复合材料及其制备方法。
为了解决技术问题,本发明首先提供了一种四氧化三锰-硫化银Janus结构纳米复合材料的制备方法,其包括如下步骤:
1)在油胺中加入乙酰丙酮锰,超声至粉末均匀分散在溶液中;在惰性气氛下进行加热反应,反应结束后,冷却至室温,离心洗涤后得到沉淀;将沉淀重新溶于二氯甲烷,超声得到澄清溶液后,加入乙醇,离心洗涤得到Mn3O4纳米颗粒沉淀,并且重新分散于环己烷中;
2)在50℃-70℃的加热条件下混合油胺和曲拉通溶液,充分搅拌后,滴入步骤1)所得的分散有四氧化三锰(Mn3O4)纳米颗粒的环己烷溶液,充分混合后滴加银氨溶液;搅拌反应30分钟到90分钟后,加入硫代乙酰胺溶液,在50℃-70℃下反应充足的时间,其中,步骤2)全过程均维持在50-70℃的加热条件下进行;
3)反应结束后加入乙醇,离心洗涤,得到Mn3O4-Ag2S纳米颗粒,即所述的四氧化三锰-硫化银Janus结构纳米材料。
作为本发明的优选方案,所述步骤1)中,油胺和乙酰丙酮锰的质量比为15:1-25:1。
作为本发明的优选方案,所述步骤1)的反应温度为180~250℃,反应时间为9~11小时。
作为本发明的优选方案,所述步骤2)中,油胺与曲拉通的摩尔比为3.6:1-4.8:1,曲拉通和银氨溶液中银离子的摩尔比为130:1,四氧化三锰纳米颗粒中的四氧化三锰与银胺溶液中银离子摩尔比为0.8-1.2:1,银氨溶液中银离子和硫代乙酰胺溶液中的硫代乙酰胺的摩尔比为1:1。
作为本发明的优选方案,所述步骤2)中,加入硫代乙酰胺溶液后,反应时间在16-30小时。
本发明还提供了一种上述方法制得的Mn3O4-Ag2S Janus纳米颗粒。
本发明还提供了一种F-127包裹的Mn3O4-Ag2S Janus纳米颗粒,其制备方法包括如下步骤:
1)将上述方法制得的Mn3O4-Ag2S Janus纳米颗粒分散在环己烷中,得到Mn3O4-Ag2SJanus纳米颗粒分散液;
2)将F-127溶于三氯甲烷中,其中,/>F-127与三氯甲烷的体积比为1:10,搅拌下滴入步骤1)所得的Mn3O4-Ag2S Janus纳米颗粒分散液,室温下搅拌;加入蒸馏水进行旋蒸,蒸发出氯仿和环己烷,得到四氧化三锰-硫化银Janus结构纳米复合材料,即/>F-127包裹的Mn3O4-Ag2S Janus纳米颗粒。
本发明还提供了一种上述方法制备的四氧化三锰-硫化银Janus结构纳米复合材料,其特征在于所述复合材料的外形均一,平均粒径为5~15nm。
本发明还提供了上述的四氧化三锰-硫化银Janus结构纳米复合材料用于制备核磁共振成像造影剂、光敏剂或抗菌剂的应用。
与现有技术相比,本发明具有的有益效果包括:
(1)本发明制备的Mn3O4-Ag2S Janus结构纳米复合材料,拥有较低的r2/r1弛豫比,在核磁共振成像造影剂方面有潜在的应用;
(2)Mn3+还在体外表现出有效的光协同作用,对肿瘤细胞有一定的光动力杀伤作用;
(3)本发明制备的Mn3O4-Ag2S Janus结构纳米复合材料,由于含有Ag2S量子点,在近红外二区有很强的吸收能力,可以有效将光转换为热能,用于消融肿瘤细胞;
(4)本发明制备的四氧化三锰-硫化银(Mn3O4-Ag2S)纳米复合材料形状规则,平均粒径为5-15nm,具有良好的分散性;
(5)本发明对实验仪器的要求低,方法简单易于操作。
附图说明
图1为本发明实施例1所得产物的透射电镜图片。
图2为本发明实施例1所得产物的透射电镜图片。
图3为本发明实施例1所得产物的动态光散射尺寸分布图。
图4为本发明实施例1所得产物的Zeta电位。
图5为本发明实施例1所得产物的X射线能谱分析(EDS)图片。
图6为本发明实施例1所得产物的X射线衍射图谱。
图7为本发明实施例1所得产物对4T1细胞的CCK-8实验结果图。
图8为本发明实施例1所得产物在T1加权下的磁共振图像。
图9为本发明实施例1所得产物的Mn浓度和纵向弛豫率的函数关系图。
图10为本发明实施例1所得产物的光热转换图像。
具体实施方式
以下实施例向本领域普通技术人员提供如何制造和评价本发明,所述实施例仅是本公开内容的示范且不圈定限制范围。尽管已经尽力确保关于数值(例如,量、温度等)的准确性,但是应当考虑一些误差和偏差。除非另外说明,否则温度是以℃为单位或者在环境温度下。
实施例一
将0.3g乙酰丙酮锰(C10H14MnO4)和7.6g的油胺混合;上述混合液体在惰性气氛下在200℃下反应10小时;等待冷却至室温后,加入10ml的二氯甲烷超声得到澄清溶液;加入40ml乙醇在9000rpm的速度离心10分钟,沉淀重新分散在10ml的环己烷里,最终得到四氧化三锰纳米颗粒;
将10ml的90%浓度的油胺和4ml生化试剂级的曲拉通溶液在50℃搅拌混合30分钟,再依次加入400μL前述制备的分散于环己烷的四氧化三锰纳米颗粒和400μL的0.02g/ml的银胺溶液,不断搅拌30分钟;随后滴加400μL的饱和硫代乙酰胺水溶液;溶液在此条件下反应24小时,待自然冷却后,用乙醇洗涤再离心3次;将纳米颗粒分散于10ml的环己烷中。用F-127对纳米复合材料进行表面改性:取1ml/>F-127,将其溶于10ml三氯甲烷(CHCl3)中,搅拌30分钟;取1ml分散于环己烷中的Mn3O4-Ag2S和1ml的三氯甲烷(CHCl3)混合,混合后逐渐滴加至上述溶液中;在室温下搅拌12小时;加入10ml蒸馏水进行旋蒸,蒸发出氯仿;得到经过/>F-127改性的纳米颗粒(/>F-127包裹的Mn3O4-Ag2S Janus纳米颗粒)。图1为本发明实施例1所得产物的透射电镜图片,图2为本发明实施例1所得产物的透射电镜图片,可以很直观的看出所制备得到的纳米粒子具有明显的Janus结构。图3为本发明实施例1所得产物的动态光散射尺寸分布的图片,可以说明所制备的最后得到的纳米粒子具有均一的尺寸分布。图4为本发明是实施例1所得产物的Zeta电位,可以说明经过/>F-127修饰的Mn3O4-Ag2S纳米颗粒电位由正值趋于负值,有利于其在生物体内的循环。图5为本发明实施例1所得产物的X射线能谱分析(EDS)图片,可以说明所制备的纳米粒子含有Mn、Ag和S四种元素。图6为本发明实施例1所得产物的X射线衍射图谱,可以说明所制备的纳米粒子有明显的Mn3O4和Ag2S的衍射峰。图8和图9分别为本发明实施例1所得产物在室温下所测的T1加权的磁共振图像和其浓度与纵向弛豫率的函数关系图,可以说明所制备的纳米粒子具有与浓度正相关的磁共振成像能力。图10为本发明实施例1所得不同浓度的产物在1.2w/cm2的1064nm激光照射下的光热转换图,可以说明所制备的纳米粒子在近红外二区有良好的吸收能力,并且可以有效的将光能转换为热能。
实施例二
将0.3g乙酰丙酮锰(C10H14MnO4)和7.6g的油胺混合;上述混合液体在惰性气氛下在200℃下反应10小时;等待冷却至室温后,加入10ml的二氯甲烷超声得到澄清溶液;加入40ml乙醇在9000rpm的速度离心10分钟,沉淀重新分散在10ml的环己烷里,最终得到四氧化三锰纳米颗粒;
将10ml油胺和4ml曲拉通溶液在50℃下搅拌混合30分钟,再依次加入800μL前述制备的分散于环己烷的四氧化三锰纳米颗粒和400μL银胺溶液,不断搅拌30分钟;随后滴加400μL的饱和硫代乙酰胺水溶液;溶液在此条件下反应24小时,待自然冷却后,用乙醇洗涤再离心3次;将纳米颗粒分散于10ml的环己烷中。用F-127对纳米复合材料进行表面改性:取1ml
F-127,将其溶于10ml三氯甲烷(CHCl3)中,搅拌30分钟;取1ml分散于环己烷中的Mn3O4-Ag2S和1ml的三氯甲烷(CHCl3)混合,混合后逐渐滴加至上述溶液中;在室温下搅拌12小时;加入10ml蒸馏水进行旋蒸,蒸发出氯仿;得到经过/>F-127改性的纳米颗粒,平均粒径为5-15nm,具有良好的分散性。
实施例三
将0.3g乙酰丙酮锰(C10H14MnO4)和7.6g的油胺混合;上述混合液体在惰性气氛下在200℃下反应10小时;等待冷却至室温后,加入10ml的二氯甲烷超声得到澄清溶液;加入40ml乙醇在9000rpm的速度离心10分钟,沉淀重新分散在10ml的环己烷里,最终得到四氧化三锰纳米颗粒;
将10mL油胺和4mL曲拉通溶液在70℃下搅拌混合30分钟,再依次加入400μL前述制备的分散于环己烷的四氧化三锰纳米颗粒和400μL银胺溶液,不断搅拌30分钟;随后滴加400μL的饱和硫代乙酰胺水溶液;溶液在此条件下反应30小时;用乙醇洗涤再离心3次;将纳米颗粒分散于10ml的环己烷中。用F-127对纳米复合材料进行表面改性:取1mlF-127,将其溶于10ml三氯甲烷(CHCl3)中,搅拌30分钟;取1ml分散于环己烷中的Mn3O4-Ag2S和1ml的三氯甲烷(CHCl3)混合,混合后逐渐滴加至上述溶液中;在室温下搅拌12小时;加入10ml蒸馏水进行旋蒸,蒸发出氯仿;得到经过/>F-127改性的纳米颗粒,平均粒径为5-15nm,具有良好的分散性。
对比例一
将0.3g乙酰丙酮锰(C10H14MnO4)和7.6g的油胺混合;上述混合液体在惰性气氛下在200℃下反应10小时;等待冷却至室温后,加入10ml的二氯甲烷超声得到澄清溶液;加入40mL乙醇在9000rpm的速度离心10分钟,沉淀重新分散在10mL的环己烷里,最终得到四氧化三锰纳米颗粒;
将10mL油胺和4mL曲拉通溶液在常温下下搅拌混合30分钟,再依次加入400μL分散于环己烷的四氧化三锰和400μL银胺溶液,不断搅拌30分钟;随后滴加400μL的饱和硫代乙酰胺水溶液;溶液在此条件下反应24小时,待自然冷却后,用乙醇洗涤再离心3次;将纳米颗粒分散于10ml的环己烷中。用F-127对纳米复合材料进行表面改性:取1mlF-127,将其溶于10ml三氯甲烷(CHCl3)中,搅拌30分钟;取1ml分散于环己烷中的Mn3O4-Ag2S和1ml的三氯甲烷(CHCl3)混合,混合后逐渐滴加至上述溶液中;在室温下搅拌12小时;加入10ml蒸馏水进行旋蒸,蒸发出氯仿;得到经过/>F-127改性的纳米颗粒。通过此对比例无法得到四氧化三锰-硫化银Janus纳米复合材料。
对比例二
将0.3g乙酰丙酮锰(C10H14MnO4)和7.6g的油胺混合;上述混合液体在惰性气氛下在200℃下反应10小时;等待冷却至室温后,加入10ml的二氯甲烷超声得到澄清溶液;加入40mL乙醇在9000rpm的速度离心10分钟,沉淀重新分散在10mL的环己烷里,最终得到四氧化三锰纳米颗粒;
将10mL油胺和4mL曲拉通溶液在50℃下搅拌混合30分钟,再依次加入400μL分散于环己烷的四氧化三锰和400μL银胺溶液,随后直接滴加400μL的饱和硫代乙酰胺水溶液;溶液在此条件下反应24小时,待自然冷却后,用乙醇洗涤再离心3次;将纳米颗粒分散于10ml的环己烷中。用F-127对纳米复合材料进行表面改性:取1ml/>F-127,将其溶于10ml三氯甲烷(CHCl3)中,搅拌30分钟;取1ml分散于环己烷中的Mn3O4-Ag2S和1ml的三氯甲烷(CHCl3)混合,混合后逐渐滴加至上述溶液中;在室温下搅拌12小时;加入10ml蒸馏水进行旋蒸,蒸发出氯仿;得到经过/>F-127改性的纳米颗粒。通过此对比例无法得到四氧化三锰-硫化银Janus纳米复合材料。
应用例
将4T1(105个细胞/孔)用适当的培养基接种到96孔板,并在37℃和5%的二氧化碳条件下保持过夜。之后,去除培养基,再用磷酸盐缓冲盐水
(PBS)清洗。在测试之前,将实施例1所得F-127改性的纳米颗粒(0、12、25、50、75和100μg/mL)处理溶解在其培养基中。每个细胞系用100μL的SBT-MET在培养基中培养24小时。通过在96孔板的每个孔中引入10μL的细胞计数试剂盒-8(CCK8)在37℃下培养2小时来评估癌症和正常细胞的增殖。完成后,使用酶标仪在450纳米处记录吸光度。图7为本发明实施例1所得产物和4T1细胞共同孵育的结果,可以说明所制备的纳米粒子具有良好的生物相容性。
最终结论:本发明的四氧化三锰-硫化银Janus结构纳米复合材料在上述浓度下具有良好的生物相容性,在该方法制备的纳米复合材料具有在癌症治疗应用中的核磁共振增强效果和作为光敏剂的潜力。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (9)
1.一种四氧化三锰-硫化银Janus结构纳米复合材料的制备方法,其特征在于,包括如下步骤:
1)在油胺中加入乙酰丙酮锰,超声至粉末均匀分散在溶液中;在惰性气氛下进行加热反应,反应结束后,冷却至室温,离心洗涤后得到沉淀;将沉淀重新溶于二氯甲烷,超声得到澄清溶液后,加入乙醇,离心洗涤得到Mn3O4纳米颗粒沉淀,并且重新分散于环己烷中;
2)在50℃-70℃的加热条件下混合油胺和曲拉通溶液,充分搅拌后,滴入步骤1)所得的分散有四氧化三锰(Mn3O4)纳米颗粒的环己烷溶液,充分混合后滴加银氨溶液;搅拌反应30分钟到90分钟后,加入硫代乙酰胺溶液,在50℃-70℃下反应充足的时间,其中,步骤2)全过程均维持在50-70℃的加热条件下进行;
3)反应结束后加入乙醇,离心洗涤,得到Mn3O4-Ag2S纳米颗粒,即所述的四氧化三锰-硫化银Janus结构纳米材料。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤1)中,油胺和乙酰丙酮锰的质量比为15:1-25:1。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤1)的反应温度为180~250℃,反应时间为9~11小时。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤2)中,油胺与曲拉通的摩尔比为3.6:1-4.8:1,曲拉通和银氨溶液中银离子的摩尔比为130:1,四氧化三锰纳米颗粒中的四氧化三锰与银胺溶液中银离子摩尔比为0.8-1.2:1,银氨溶液中银离子和硫代乙酰胺溶液中的硫代乙酰胺的摩尔比为1:1。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤2)中,加入硫代乙酰胺溶液后,反应时间在16-30小时。
6.权利要求1-5任一项所述方法制得的Mn3O4-Ag2S Janus纳米颗粒。
7.一种PF127包裹的Mn3O4-Ag2S Janus纳米颗粒,其特征在于,其制备方法包括如下步骤:
1)将权利要求1-5任一项所述方法制得的Mn3O4-Ag2S Janus纳米颗粒分散在环己烷中,得到Mn3O4-Ag2S Janus纳米颗粒分散液;
2)将F-127完全溶于三氯甲烷中,其中,/>F-127与三氯甲烷的质量比为1:80-120,搅拌下滴入步骤1)所得的Mn3O4-Ag2S Janus纳米颗粒分散液,其中Mn3O4-Ag2SJanus纳米颗粒与/>F-127的质量比为1:103,室温下搅拌;加入蒸馏水进行旋蒸,蒸发出氯仿和环己烷,得到四氧化三锰-硫化银Janus结构纳米复合材料,即/>F-127包裹的Mn3O4-Ag2S Janus纳米颗粒。
8.根据权利要求7所述的PF127包裹的Mn3O4-Ag2S Janus纳米颗粒,其特征在于所述复合材料的外形均一,平均粒径为5~15nm。
9.权利要求7所述的PF127包裹的Mn3O4-Ag2S Janus纳米颗粒用于制备核磁共振成像造影剂、光敏剂或抗菌剂的应用。
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