纳米磁颗粒的表面修饰及其在生物学上的应用
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摘要
纳米磁颗粒是一种在生物医学领域有着重大应用价值的新型材料。纳米磁颗粒的表面修饰对其实际应用有着极其重要的影响。总体上说,表面修饰起到如下三个方面的作用:(1)控制纳米磁颗粒的尺寸与形貌;(2)赋予纳米磁颗粒合适的胶体稳定性;(3)进行纳米磁颗粒的生物学功能化。表面修饰在很大程度上决定了纳米磁颗粒的应用价值。本课题以基因操纵与转染为应用目的,制备了小分子,高分子,和无机纳米颗粒等多种材料修饰的纳米磁性颗粒。首先,我们合成了羧甲基葡聚糖,氨基硅烷,甜菜碱,柠檬酸,聚乙烯亚胺等一系列材料修饰的磁颗粒,并将它们用于增强绿色荧光蛋白质粒(GFP)转染NIH-3T3细胞。我们还合成了磷酸钙-四氧化三铁复合物,应用于基因的可控运输和转染。此外,我们还分别制备了稳定结合的金磁复合物以及能够可控解离的金磁双硫键组装体。最后,我们将温敏高分子-紫杉醇复合物包覆在纳米磁颗粒上,研究了交变磁场控制胶体稳定性的可行性。经过如上所述的各项工作,得出结论如下:
(1)在聚乙烯亚胺转染细胞的实验中,加入纳米磁颗粒并在磁板上静置15分钟,可以大大增强最终的基因表达效率,同时减轻受体细胞在转染过程中所受的伤害。然而只有团聚尺寸较小(Dh < 100 nm)的聚乙烯亚胺包覆纳米磁颗粒才有明显的磁转染功效,过大的团聚体甚至会抑制基因的表达;
(2)在磁转染中并不一定需要聚乙烯亚胺包覆的纳米磁颗粒,只要磁颗粒的表面带有足够的电荷,无论是正电荷还是负电荷,均能在磁场下增强基因表达的效率;
(3)新型的磷酸钙-四氧化三铁复合纳米材料,在钙离子和碳酸根离子的调节下,可以成为一种高效、载带/卸载完全可控的DNA载体。在磁场增强的条件下,它在体外实验中能够高效地转染质粒DNA进入COS-7细胞;
(4)利用聚乙烯亚胺包覆的纳米磁颗粒直接还原氯金酸,可以得到一种新型的金磁复合物。它对巯基类分子有着特异性的吸附能力,而对氨基类分子则几乎不吸收;
(5)N-琥珀酰亚-3-(2-吡啶基二硫)丙酸酯(SPDP)可以将氨基化的金颗粒组装到巯基化的磁微球表面。这种复合物结构遇到巯基乙醇即可发生解离,金颗粒从磁微球上脱离,重新分散到溶液中。因此,这里制备的金磁双硫键复合物是一种“可切割”的二元纳米组装材料;
(6)交变磁场可以控制温敏性纳米磁颗粒的胶体稳定性以及磁响应性。
Magnetic nanoparticles have many important biomedical applications. The surface modification to the particles can dramatically influence their application values. Generally speaking, surface modification has the following three functions: (1) controlling the size and morphology of magnetic nanoparticles; (2) adjusting the colloidal stabilities of the particles; (3) delivering bio-functions to magnetic nanoparticles. In this PhD program, we have prepared magnetic naoparticles modified by various small molecules, polymers, and inorganic nanoparticlers. First,we have prepared dextran, aminosilane, citric acid, betaine, and PEI modified particles, and used them to enhance transfecting plasmid DNA into cells in vitro. Then we prepared calcium phosphate-magnetite hybrid materials to deliver and transfect DNA. We also combined gold nanoparticles onto magnetic nanoparticles to make gold-magnetic hybrid materials. We have prepared both stably combined and“cleavable”complexes. Finally, we coated a thermo-sensitive polymer onto the magnetic particles and then used alternating magnetic field to control the aggregation of such particles. Based on these work, we have the following main points of results:
(1) The aggregation size of magnetic nanoparticles has dramatic effect on their performances in magnetofection. Only those weakly aggregated particles can successfully enhance gene transfection.
(2) If the particles have sufficiently high surface potentials, either positive or negative, they are capable of enhancing the gene delivery of PEI. In magnetofection, the used magnetic nanoparticles don’t have to be coated with functional transfection agents. Instead, surface charged particles can also work well. The preparation of magnetic nanoparticles and gene vector can be separated in a magnetofection study.
(3) A novel calcium phosphate-magnetite composite nanomaterial has been prepared. In solution of CaCl_2, such material is able to load a large amount of DNA by co-assembling into complex microspheres. The microspheres are broken in the solution of CO_3~(2-) and release the loaded DNA, which is still available for PCR amplification. Enhanced by magnetic force, the CaP-M is able to effectively transfect plasmids into COS-7 cells in vitro.
(4) Au/PEI/Fe_3O_4 nanocomposite has been prepared via in-situ reducing dissolved HAuCl4 by PEI coated magnetic nanoparticles. The composite is capable of specifically capturing biomolecules with thiol groups.
(5) We have designed and prepared gold-magnetic composites where two particles are combined by disulfide bonds. The composites can be cleaved byβ-mercaptoethanol in their dispersions, the gold and magnetic beads are thus separated.
(6) The colloidal stabilities and magnetic responses of thermosensitive polymer coated magnetic nanoparticlers can be controlled by a remote alternating magnetic field.
(1)在聚乙烯亚胺转染细胞的实验中,加入纳米磁颗粒并在磁板上静置15分钟,可以大大增强最终的基因表达效率,同时减轻受体细胞在转染过程中所受的伤害。然而只有团聚尺寸较小(Dh < 100 nm)的聚乙烯亚胺包覆纳米磁颗粒才有明显的磁转染功效,过大的团聚体甚至会抑制基因的表达;
(2)在磁转染中并不一定需要聚乙烯亚胺包覆的纳米磁颗粒,只要磁颗粒的表面带有足够的电荷,无论是正电荷还是负电荷,均能在磁场下增强基因表达的效率;
(3)新型的磷酸钙-四氧化三铁复合纳米材料,在钙离子和碳酸根离子的调节下,可以成为一种高效、载带/卸载完全可控的DNA载体。在磁场增强的条件下,它在体外实验中能够高效地转染质粒DNA进入COS-7细胞;
(4)利用聚乙烯亚胺包覆的纳米磁颗粒直接还原氯金酸,可以得到一种新型的金磁复合物。它对巯基类分子有着特异性的吸附能力,而对氨基类分子则几乎不吸收;
(5)N-琥珀酰亚-3-(2-吡啶基二硫)丙酸酯(SPDP)可以将氨基化的金颗粒组装到巯基化的磁微球表面。这种复合物结构遇到巯基乙醇即可发生解离,金颗粒从磁微球上脱离,重新分散到溶液中。因此,这里制备的金磁双硫键复合物是一种“可切割”的二元纳米组装材料;
(6)交变磁场可以控制温敏性纳米磁颗粒的胶体稳定性以及磁响应性。
Magnetic nanoparticles have many important biomedical applications. The surface modification to the particles can dramatically influence their application values. Generally speaking, surface modification has the following three functions: (1) controlling the size and morphology of magnetic nanoparticles; (2) adjusting the colloidal stabilities of the particles; (3) delivering bio-functions to magnetic nanoparticles. In this PhD program, we have prepared magnetic naoparticles modified by various small molecules, polymers, and inorganic nanoparticlers. First,we have prepared dextran, aminosilane, citric acid, betaine, and PEI modified particles, and used them to enhance transfecting plasmid DNA into cells in vitro. Then we prepared calcium phosphate-magnetite hybrid materials to deliver and transfect DNA. We also combined gold nanoparticles onto magnetic nanoparticles to make gold-magnetic hybrid materials. We have prepared both stably combined and“cleavable”complexes. Finally, we coated a thermo-sensitive polymer onto the magnetic particles and then used alternating magnetic field to control the aggregation of such particles. Based on these work, we have the following main points of results:
(1) The aggregation size of magnetic nanoparticles has dramatic effect on their performances in magnetofection. Only those weakly aggregated particles can successfully enhance gene transfection.
(2) If the particles have sufficiently high surface potentials, either positive or negative, they are capable of enhancing the gene delivery of PEI. In magnetofection, the used magnetic nanoparticles don’t have to be coated with functional transfection agents. Instead, surface charged particles can also work well. The preparation of magnetic nanoparticles and gene vector can be separated in a magnetofection study.
(3) A novel calcium phosphate-magnetite composite nanomaterial has been prepared. In solution of CaCl_2, such material is able to load a large amount of DNA by co-assembling into complex microspheres. The microspheres are broken in the solution of CO_3~(2-) and release the loaded DNA, which is still available for PCR amplification. Enhanced by magnetic force, the CaP-M is able to effectively transfect plasmids into COS-7 cells in vitro.
(4) Au/PEI/Fe_3O_4 nanocomposite has been prepared via in-situ reducing dissolved HAuCl4 by PEI coated magnetic nanoparticles. The composite is capable of specifically capturing biomolecules with thiol groups.
(5) We have designed and prepared gold-magnetic composites where two particles are combined by disulfide bonds. The composites can be cleaved byβ-mercaptoethanol in their dispersions, the gold and magnetic beads are thus separated.
(6) The colloidal stabilities and magnetic responses of thermosensitive polymer coated magnetic nanoparticlers can be controlled by a remote alternating magnetic field.
引文
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