荧光和多孔胶体微粒的制备及与细胞相互作用的研究
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摘要
在生物学和医学的研究领域中,各种功能胶体微粒作为荧光探针以及药物或基因的载体,其中的一个核心问题是细胞与胶体微粒之间的相互作用,在生物分析、检测以及药物传输和基因治疗等领域得到广泛的应用。
本文首先尝试了制备具有荧光特性的胶体微粒。利用水相合成法成功得到水溶性的以3-巯基丙酸为稳定剂(MPA)的CdTe量子点(QDs),并对制备条件进行了优化。结果表明,当前驱体溶液pH值为9.0,各原料的投料摩尔比满足Cd:Te:MPA=1:0.2:2.4,且[Cd~(2+)]=1.25mM,在高温高压下回流时可以得到量子产率超过40%的高质量水溶性MPA-CdTe QDs。
利用共沉淀法将水溶性CdTe量子点包埋在无机盐中,制备了掺杂MPA-CdTeQDs的CaCO_3胶体微粒,并讨论了制备条件如溶液的pH值、QDs的加入量以及原料的投料顺序对得到的复合胶体粒子的粒径、对量子点的包埋率以及相对发光效率的影响。其中混合溶液的pH值与原料的投料顺序对粒子的粒径影响较大,而加入的QDs的量会影响包埋率以及相对发光效率。当量子点先与硝酸钙溶液混合且混合溶液的pH值大于7.0时可以得到较小的复合微粒;随着反应原料中QDs量的增加,尽管QDs的包埋效率及其发光效率都有明显下降,但是总的发光强度有所增加。MPA-CdTe QDs被包入碳酸钙粒子之后,其存贮稳定性和光学稳定性都有一定程度的提高。当CaCO_3(CdTe)复合微粒在细胞培养液中存贮6天或者在24h紫外灯的连续照射下能够保持其荧光强度基本不变。CaCO_3(CdTe)粒子能够被HepG2细胞胞吞,在与细胞共培养24h之后可以进入细胞的溶酶体中。同时在溶液中CdTe的浓度相同的条件下,CaCO_3(CdTe)复合微粒对细胞的毒性要远远小于CdTe QDs。
利用St(o|¨)ber方法合成了四种粒径的单分散荧光二氧化硅纳米粒子,分别用透射电镜、荧光光谱、动态光散射和zeta电位分析仪对其形貌和表面性质进行了表征。在透射电镜下这四组纳米粒子的平均粒径分别为60.3nm(S60)、177.8nm(S180)、368.6nm($370)和592.1nm(S600)。当它们被分散在DMEM(Dulbecco'sModified Eagles Medium)/10%FBS(fetal bovine serum)溶液中时,粒径较小的粒子如S60更容易发生团聚,并且它们的zeta电位值相近,约为-5mV。以这四种粒径的荧光二氧化硅纳米粒子为模型,讨论了粒子的粒径对细胞的胞吞、胞吐、粒子进入细胞的途径、在细胞中的分布以及它们的细胞毒性的影响。实验结果表明细胞对二氧化硅纳米粒子的胞吞和胞吐具有粒径依赖性。在40μg/ml的浓度下,HepG2细胞、成纤维细胞以及Ana-1巨噬细胞对S370的胞吞量最大。而HepG2细胞吐出二氧化硅纳米粒子的难度与粒子的粒径成正关系。二氧化硅纳米粒子进入细胞的途径和它们在细胞中的分布不受粒子粒径的影响。细胞对二氧化硅纳米粒子的胞吞具有能量依赖性,不同大小的二氧化硅纳米粒子都是通过网格蛋白介导的胞吞途径进入HepG2细胞中的,并且分布在细胞膜、细胞质和溶酶体中。不同粒径的二氧化硅纳米粒子对HepG2细胞的毒性都很小,特别是S60在较低的浓度时(280μg/10~5个细胞)还能在一定程度上提高细胞的活性。二氧化硅纳米粒子的细胞毒性仍然呈现出粒径依赖性,但是它们对细胞周期的影响不具有粒径依赖性。
以多孔碳酸钙微粒为模板,制备了具有多孔结构的二氧化硅胶体微粒。分别用透射电镜、扫描电镜、X射线能谱、红外光谱、X-射线衍射、热重分析、氮气的吸附一脱吸附方法以及zeta电位分析仪对其组成、表面形态以及内部结构进行了表征。得到的多孔二氧化硅胶体微球在干态下是3～5μm的多孔微球,比表面积达到367.3 m~2/g,并且粒子中孔的大小是多分散的。反应体系中正硅酸乙酯(TEOS)的浓度对产物的结构影响较大。当TEOS的浓度较大时,只能得到空心的二氧化硅微球而非多孔结构的二氧化硅微球。此外,当碳酸钙模板表面吸附一层聚二烯炳基二甲基铵盐酸盐(PDADMAC)时,制得的多孔二氧化硅胶体微球更易于保持碳酸钙模板的形状。此外,以掺杂聚苯乙烯磺酸钠(PSS)的碳酸钙微粒为模板,可以制得空心的二氧化硅微囊,与干态下的聚电解质微胶囊的形态极为相似。多孔二氧化硅胶体微球在水溶液中带负电,能够使带正电的葡聚糖(TRITC-dextran)在其内部沉积,而对于带负电的葡聚糖(FITC-dextran),仅在葡聚糖的分子量较小时,才能在多孔二氧化硅胶体微球中部分沉积。制得的多孔二氧化硅胶体微球可以吸附活性辣根过氧化物酶(HRP)。增加溶液中HRP的浓度可以明显增加多孔二氧化硅胶体微球对HRP的吸附量,略微增加HRP的吸附效率,但是被吸附的HRP的酶活性有一定程度的损失,且被吸附的HRP的剩余活性随溶液中HRP浓度的增加而大幅度下降。
Along with the fast development of nanotechnology and nanomedicine,the colloidal particles are usually integrated with multifunction such as tailored wettability,specific targeting,imaging and controlled release of the desired substances to be used as carriers for drug control release,cancer and gene therapy,disease diagnosis and bio-imaging via the way of intracellular uptake and delivery.
First,we synthesized the water-soluble 3-mercaptopropyl acid(MPA) capped CdTe nanocrystals and optimized the preparation conditions.Results showed that when the pH of precursor was fixed at 9.0 and the molar ratio of Cd:Te:MPA is 1:0.2: 2.4 with a Cd~(2+) concentration of 1.25mM,the high quality CdTe nanocrystals could be obtained under a high pressure and temperature condition with a quantum yield up to more than 40%.
The QDs could also be incorporated into inorganic colloidal particles.The MPA-CdTe quantum dots(QDs) were embedded into CaCO_3 microparticles with a size of 1.4～4.4μm by addition of the QDs into Ca(NO_3)_2 solution during a mineralization process.Compared to the parent QDs,about 1/7～/1/4 photoluminescence efficiency of the embedded QDs was preserved,enabling the CaCO_3(CdTe) particles visible under UV irradiation.The structure and morphology of the CaCO_3(CdTe) particles were characterized by X-ray diffraction(XRD),UV-vis spectroscopy,scanning electron microscopy(SEM),transmission electron microscopy (TEM) and confocal laser scanning microscopy(CLSM).Protected by the CaCO_3 particles,the QDs in the composites were more stable against long term storage,UV irradiation and cell culture medium containing serum.The CaCO_3(CdTe) particles could be internalized into live ceils,human liver cancer cells(HepG2) cells,for example,and most of which distributed in the lysosomes as revealed by confocal microscopy.Also the CaCO_3(CdTe) particles had low cytotoxicity in comparison with the parent CdTe QDs.
Next,fluorescent silica nanoparticles with four different diameters were prepared by the classical St(o|¨)ber sol-gel method.When these silica particles were suspended into the DMEM(Dulbecco's Modified Eagles Medium)/10%FBS(fetal bovine serum) solution,some extent aggregation appeared especially for the smaller ones.Their surfaces showed a similar zeta potential of about -5mV.Cellular uptake of the silica nanoparticles with different diameters was carefully studied.The results revealed that the cellular uptake of the silica nanoparticles was dependent on particle concentration, co-culture time and cell types.While in the same condition,the cells had larger uptake amount of S370(the diameter of the particles under TEM is 368.6nm) under a 40μg/ml particle concentration.The exocytosis which is the opposite process of endocytosis was also size dependant.The silica nanoparticles could be gradually cleaned out from the cells during a 12h incubation after a 12h endocytosis,finding a higher exocytosed efficiency for smaller particles.The particles in the cells mainly distributed in the cytoplasm,lysosomes and onthe cell membranes as revealed by CLSM(confocal laser scanning microscopy).The morphology of the cells after their co-culture with particles was observed by SEM showing an aggregation state on the surface of the cell membranes for smaller ones.The silica nanoparticles were internalized into the cells through a clathrin-mediated endocytosis pathway which was proved by an obvious decrease of the cellular uptake after a low temperature incubation,NaN_3,sucrose or amandatine-HCl treatments.While the genestein treatment could not influence the cellular uptake,and the result indicated a caveolae independent endocytosis for the silica nanoparticles.The impact of silica nanoparticles on the HepG2 cells was assayed in terms of cytotoxicity and cell cycle profile.The cytotoxicity was in a low extent even at a high particle concentration up to 1000μg/10,000 cells,especially for the smaller particles.The cell cycle analysis showed that the nanoparticle-treatment can not influence the cell proliferation at a concentration of 80μg/ml for 24h co-culture.
At last we synthesized porous silica particles using a CaCO_3 particle template. The porous silica particles had a spherical shape with diameters of 3～5μm and a porous structure under a dry state.Their relative surface area reached to 367.3 m~2/g with multiscale pores.The concentration of TEOS(tetraethyl orthosilicate) in the preparation system showed an apparent influence on the structure of the products. Under a relative high concentration of TEOS,hollow capsules were obtained.When a PDADMAC(poly(diallyl-dimethyl-ammoniumchloride)) layer was adsorbed on the pore surface of the CaCO_3 particles in prior,more solid porous silica particles were obtained and kept the shape of the template better.The porous silica colloidal particles showed a negatively charged surface in water.Thus,TRITC-dextran (tetramethylrhodamineisothiocyanate) which is positively charged in water could be deposited into the pores.The porous silica colloidal particles could adsorb active enzymes,too.While more HRP(horseradish peroxidase) was deposited with the increase of the HRP concentration,its bioactivity was decreased.Moreover,the PSS (polystyrene sulfonate) doped CaCO_3 particles could also be used as template for the growth of silica particles on their surface.The silica colloidal particles were hollow with only a thin wall and showed a similar morphology to polyelectrolyte microcapsules under a dry state.
本文首先尝试了制备具有荧光特性的胶体微粒。利用水相合成法成功得到水溶性的以3-巯基丙酸为稳定剂(MPA)的CdTe量子点(QDs),并对制备条件进行了优化。结果表明,当前驱体溶液pH值为9.0,各原料的投料摩尔比满足Cd:Te:MPA=1:0.2:2.4,且[Cd~(2+)]=1.25mM,在高温高压下回流时可以得到量子产率超过40%的高质量水溶性MPA-CdTe QDs。
利用共沉淀法将水溶性CdTe量子点包埋在无机盐中,制备了掺杂MPA-CdTeQDs的CaCO_3胶体微粒,并讨论了制备条件如溶液的pH值、QDs的加入量以及原料的投料顺序对得到的复合胶体粒子的粒径、对量子点的包埋率以及相对发光效率的影响。其中混合溶液的pH值与原料的投料顺序对粒子的粒径影响较大,而加入的QDs的量会影响包埋率以及相对发光效率。当量子点先与硝酸钙溶液混合且混合溶液的pH值大于7.0时可以得到较小的复合微粒;随着反应原料中QDs量的增加,尽管QDs的包埋效率及其发光效率都有明显下降,但是总的发光强度有所增加。MPA-CdTe QDs被包入碳酸钙粒子之后,其存贮稳定性和光学稳定性都有一定程度的提高。当CaCO_3(CdTe)复合微粒在细胞培养液中存贮6天或者在24h紫外灯的连续照射下能够保持其荧光强度基本不变。CaCO_3(CdTe)粒子能够被HepG2细胞胞吞,在与细胞共培养24h之后可以进入细胞的溶酶体中。同时在溶液中CdTe的浓度相同的条件下,CaCO_3(CdTe)复合微粒对细胞的毒性要远远小于CdTe QDs。
利用St(o|¨)ber方法合成了四种粒径的单分散荧光二氧化硅纳米粒子,分别用透射电镜、荧光光谱、动态光散射和zeta电位分析仪对其形貌和表面性质进行了表征。在透射电镜下这四组纳米粒子的平均粒径分别为60.3nm(S60)、177.8nm(S180)、368.6nm($370)和592.1nm(S600)。当它们被分散在DMEM(Dulbecco'sModified Eagles Medium)/10%FBS(fetal bovine serum)溶液中时,粒径较小的粒子如S60更容易发生团聚,并且它们的zeta电位值相近,约为-5mV。以这四种粒径的荧光二氧化硅纳米粒子为模型,讨论了粒子的粒径对细胞的胞吞、胞吐、粒子进入细胞的途径、在细胞中的分布以及它们的细胞毒性的影响。实验结果表明细胞对二氧化硅纳米粒子的胞吞和胞吐具有粒径依赖性。在40μg/ml的浓度下,HepG2细胞、成纤维细胞以及Ana-1巨噬细胞对S370的胞吞量最大。而HepG2细胞吐出二氧化硅纳米粒子的难度与粒子的粒径成正关系。二氧化硅纳米粒子进入细胞的途径和它们在细胞中的分布不受粒子粒径的影响。细胞对二氧化硅纳米粒子的胞吞具有能量依赖性,不同大小的二氧化硅纳米粒子都是通过网格蛋白介导的胞吞途径进入HepG2细胞中的,并且分布在细胞膜、细胞质和溶酶体中。不同粒径的二氧化硅纳米粒子对HepG2细胞的毒性都很小,特别是S60在较低的浓度时(280μg/10~5个细胞)还能在一定程度上提高细胞的活性。二氧化硅纳米粒子的细胞毒性仍然呈现出粒径依赖性,但是它们对细胞周期的影响不具有粒径依赖性。
以多孔碳酸钙微粒为模板,制备了具有多孔结构的二氧化硅胶体微粒。分别用透射电镜、扫描电镜、X射线能谱、红外光谱、X-射线衍射、热重分析、氮气的吸附一脱吸附方法以及zeta电位分析仪对其组成、表面形态以及内部结构进行了表征。得到的多孔二氧化硅胶体微球在干态下是3～5μm的多孔微球,比表面积达到367.3 m~2/g,并且粒子中孔的大小是多分散的。反应体系中正硅酸乙酯(TEOS)的浓度对产物的结构影响较大。当TEOS的浓度较大时,只能得到空心的二氧化硅微球而非多孔结构的二氧化硅微球。此外,当碳酸钙模板表面吸附一层聚二烯炳基二甲基铵盐酸盐(PDADMAC)时,制得的多孔二氧化硅胶体微球更易于保持碳酸钙模板的形状。此外,以掺杂聚苯乙烯磺酸钠(PSS)的碳酸钙微粒为模板,可以制得空心的二氧化硅微囊,与干态下的聚电解质微胶囊的形态极为相似。多孔二氧化硅胶体微球在水溶液中带负电,能够使带正电的葡聚糖(TRITC-dextran)在其内部沉积,而对于带负电的葡聚糖(FITC-dextran),仅在葡聚糖的分子量较小时,才能在多孔二氧化硅胶体微球中部分沉积。制得的多孔二氧化硅胶体微球可以吸附活性辣根过氧化物酶(HRP)。增加溶液中HRP的浓度可以明显增加多孔二氧化硅胶体微球对HRP的吸附量,略微增加HRP的吸附效率,但是被吸附的HRP的酶活性有一定程度的损失,且被吸附的HRP的剩余活性随溶液中HRP浓度的增加而大幅度下降。
Along with the fast development of nanotechnology and nanomedicine,the colloidal particles are usually integrated with multifunction such as tailored wettability,specific targeting,imaging and controlled release of the desired substances to be used as carriers for drug control release,cancer and gene therapy,disease diagnosis and bio-imaging via the way of intracellular uptake and delivery.
First,we synthesized the water-soluble 3-mercaptopropyl acid(MPA) capped CdTe nanocrystals and optimized the preparation conditions.Results showed that when the pH of precursor was fixed at 9.0 and the molar ratio of Cd:Te:MPA is 1:0.2: 2.4 with a Cd~(2+) concentration of 1.25mM,the high quality CdTe nanocrystals could be obtained under a high pressure and temperature condition with a quantum yield up to more than 40%.
The QDs could also be incorporated into inorganic colloidal particles.The MPA-CdTe quantum dots(QDs) were embedded into CaCO_3 microparticles with a size of 1.4～4.4μm by addition of the QDs into Ca(NO_3)_2 solution during a mineralization process.Compared to the parent QDs,about 1/7～/1/4 photoluminescence efficiency of the embedded QDs was preserved,enabling the CaCO_3(CdTe) particles visible under UV irradiation.The structure and morphology of the CaCO_3(CdTe) particles were characterized by X-ray diffraction(XRD),UV-vis spectroscopy,scanning electron microscopy(SEM),transmission electron microscopy (TEM) and confocal laser scanning microscopy(CLSM).Protected by the CaCO_3 particles,the QDs in the composites were more stable against long term storage,UV irradiation and cell culture medium containing serum.The CaCO_3(CdTe) particles could be internalized into live ceils,human liver cancer cells(HepG2) cells,for example,and most of which distributed in the lysosomes as revealed by confocal microscopy.Also the CaCO_3(CdTe) particles had low cytotoxicity in comparison with the parent CdTe QDs.
Next,fluorescent silica nanoparticles with four different diameters were prepared by the classical St(o|¨)ber sol-gel method.When these silica particles were suspended into the DMEM(Dulbecco's Modified Eagles Medium)/10%FBS(fetal bovine serum) solution,some extent aggregation appeared especially for the smaller ones.Their surfaces showed a similar zeta potential of about -5mV.Cellular uptake of the silica nanoparticles with different diameters was carefully studied.The results revealed that the cellular uptake of the silica nanoparticles was dependent on particle concentration, co-culture time and cell types.While in the same condition,the cells had larger uptake amount of S370(the diameter of the particles under TEM is 368.6nm) under a 40μg/ml particle concentration.The exocytosis which is the opposite process of endocytosis was also size dependant.The silica nanoparticles could be gradually cleaned out from the cells during a 12h incubation after a 12h endocytosis,finding a higher exocytosed efficiency for smaller particles.The particles in the cells mainly distributed in the cytoplasm,lysosomes and onthe cell membranes as revealed by CLSM(confocal laser scanning microscopy).The morphology of the cells after their co-culture with particles was observed by SEM showing an aggregation state on the surface of the cell membranes for smaller ones.The silica nanoparticles were internalized into the cells through a clathrin-mediated endocytosis pathway which was proved by an obvious decrease of the cellular uptake after a low temperature incubation,NaN_3,sucrose or amandatine-HCl treatments.While the genestein treatment could not influence the cellular uptake,and the result indicated a caveolae independent endocytosis for the silica nanoparticles.The impact of silica nanoparticles on the HepG2 cells was assayed in terms of cytotoxicity and cell cycle profile.The cytotoxicity was in a low extent even at a high particle concentration up to 1000μg/10,000 cells,especially for the smaller particles.The cell cycle analysis showed that the nanoparticle-treatment can not influence the cell proliferation at a concentration of 80μg/ml for 24h co-culture.
At last we synthesized porous silica particles using a CaCO_3 particle template. The porous silica particles had a spherical shape with diameters of 3～5μm and a porous structure under a dry state.Their relative surface area reached to 367.3 m~2/g with multiscale pores.The concentration of TEOS(tetraethyl orthosilicate) in the preparation system showed an apparent influence on the structure of the products. Under a relative high concentration of TEOS,hollow capsules were obtained.When a PDADMAC(poly(diallyl-dimethyl-ammoniumchloride)) layer was adsorbed on the pore surface of the CaCO_3 particles in prior,more solid porous silica particles were obtained and kept the shape of the template better.The porous silica colloidal particles showed a negatively charged surface in water.Thus,TRITC-dextran (tetramethylrhodamineisothiocyanate) which is positively charged in water could be deposited into the pores.The porous silica colloidal particles could adsorb active enzymes,too.While more HRP(horseradish peroxidase) was deposited with the increase of the HRP concentration,its bioactivity was decreased.Moreover,the PSS (polystyrene sulfonate) doped CaCO_3 particles could also be used as template for the growth of silica particles on their surface.The silica colloidal particles were hollow with only a thin wall and showed a similar morphology to polyelectrolyte microcapsules under a dry state.
引文
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