微乳液滴模板法构筑有序多孔膜及其在生物体系中的应用
摘要
生命活性物质如蛋白质和细胞的图案化结构由于在组织工程、细胞行为研究以及生物医药设备等领域的重要应用而引起了研究者们的广泛兴趣。在制备生命活性物质图案化结构的各种方法中，自组装的方法更为简便，尤其是以水滴为模板的自组装方法由于模板的廉价、无污染、易获得和易除去等优点引起了人们的普遍关注。
本论文中，以水蒸气模板法为借鉴，以简化生命活性物质图案化结构制备过程为目的，发展了一种新颖的制备有序多孔结构的方法，利用这种方法，我们在制备得到有序蜂窝状多孔膜的同时获得了生命活性物质的图案化结构，而且也对此体系中的生命活性物质进行了进一步的研究。论文的研究内容主要分为以下三个部分：1、发展一种以微乳液为成膜体系，微乳液中的水滴为模板制备有序蜂窝状多孔膜的普适方法–微乳液滴模板法，并且研究此方法的各种影响因素，基于对各种影响因素的分析和讨论，提出此方法制备有序多孔膜的机理；2、由于在微乳液滴模板法中，作为模板的水滴来自于成膜体系而非从外界引入，所以通过将水溶性的蛋白质事先溶解在水相，可将蛋白质引入到制得多孔膜的孔洞内部，进而制备得到与孔洞排列相近的蛋白阵列，实现蛋白图案化结构的一步制备；3、以纯水作为水相，可制备孔洞内壁负载有正电荷表面活性剂的有序多孔结构，以此表面活性剂层为基础，可以组装与细胞带相反电荷的聚电解质，进而实现细胞在孔洞内部的吸附，由于选用细胞的大小与孔洞尺寸相近，所以可制备得到每个孔洞内只有一个细胞存在的单细胞阵列，进一步的，可研究细胞在孔洞内的行为，当组装的聚电解质为DNA时，还可实现细胞在孔洞内部的原位转染，进而研究组装层数对转染效率的影响。
以上研究工作为有序多孔结构的制备提供了一种新的方法，同时简化了水溶性生物材料的图案化结构制备过程，此外，以上研究工作的结果对细胞行为的研究也具有重要的应用价值。
Micropattern of biomaterials has attracted considerable attention due to their usein tissue engineering, cell investigation and biological detection. Various methodshave been developed for the fabrication of biomaterial patterns, top-down techniquesare the widely used ones. These methods are feasible, but the expensive equipmentand complex operation steps make them inconvenient for their application in manyareas. Self-assembly methods are more convenient compared to the top-downtechniques. Recently, breath figure method is attractive due to the use of cheap andnontoxic water droplets as template. However, the introduction of functional materialsalways realized by doping in organic solution or through complex introduction steps,which limit the functionalization with water soluble functional materials especiallywith brittle water soluble materials such as protein. Therefore, it is important todevelop a more simple method to achieve the fabrication of biomaterial pattern.
In this dissertation, we develop a microemlsion method which also uses thecheap and nontoxic water droplets as template. We realize the convenient fabricationof biomaterial pattern through this microemulsion method. Using thehoneycomb-patterned porous film prepared by this microemulsion method as atemplate, ordered cell pattern can be obtained. Furthermore, the activities of cells onthe honeycomb-patterned porous film can be investigated.
First, to simplify the introduction process of the water soluble materials, wedevelop a microemulsion method which also uses water droplets as template for thefabrication of ordered porous film. We use the microemulsion as casting solution,water droplets which come from the water phase of the microemulsion as template forthe honeycomb-patterned porous structure in this microemulsion method. Through thesize comparison of primary water droplets in micoemulsion and the pores in porousfilm, we found that the pores are much larger than the primary water droplets, thus thepores were formed by the fusion of primary water droplets in microemulsion.Furthermore, we also investigate the influence of solvent, polymer, surfactant, volumeratio of water phase and casting volume on the formation and order of the porous film.The surfactant distribute at oil-water interface to stabilize the water droplets due totheir amphiphilicity, thus the addition of surfactant is critical for the formation ofordered porous structure. The order and pore size of the porous film can be adjustedby varying the concentration of surfactant. In addition, solvent, polymer, volume ratioof water and casting volume can also influence the order and pore size of the porousfilm through affecting the evaporation speed of solvent and the stability of the waterdroplets. The investigations of these factors also confirm the existence of water fusionduring the formation of porous film. We propose a possible mechanism for thefabrication of ordered honeycomb-patterned porous film through microemulsionmethod based on the analysis of all the results, which is valuable for thefunctionalization of the porous film.
Second, in the microemulsion method, the water droplets come from water phaseof the microemulsion, therefore, if we add the water soluble functional material intowater phase beforehand, it will be exist in the water droplets during the formation ofordered porous film and then fall down to the bottom of the pores with theevaporation of water, thus form the ordered porous film which was functionalized bythis water soluble material. Based on the above analysis, we dissolved the protein intowater phase aforehand, and then use this protein aqueous solution as a water phase toprepare the protein contained microemulsion. After casting this protein contained microemulsion onto proper substrate, protein contained porous film can be obtained.Through the confocal laser scanning microscopy characterization after proteinlabeling with fluorescent dye, we found that most of the protein locate at the bottomof the pores, thus form the similar pattern with the pores. Therefore, we can preparethe ordered porous film and protein pattern at the same time. Furthermore, thepatterned protein can interact with the molecular that can specifically recognize it,thus we can distinguish different kinds of protein using this protein pattern.
Third, during the formation of ordered porous film, the surfactant locate at theoil-water interface, so it will be exist at the inner surface of the pores after the filmformation. In our system, we choose the positively charged DDAB as a surfactant,therefore, we can use this DDAB layer as a substrate for polyelectrolyte assemblybased on electrostatic interaction. After the assembly of positively chargedpolyelectrolyte on the inner surface of the pores, we can use this polyelectrolyte layeras a substrate to achieve the adhesion of negatively charged cell, and thus form thecell pattern similar to the array of the ordered pores. Here, we select the yeast cellswhich have the similar size to the patterned pores. Therefore, we can prepare orderedcell pattern with single cell in one pore, which is important for the investigation ofsingle cell. And the viability of the yeast cells retained through the FDA test.Furthermore, the yeast cells can grow larger till form the similar morphology with thepores after culturing for12hours, but their proliferation was inhibited due to the limitof the pores. The grown cells can proliferate again after releasing from the poresthrough immersing into sodium chloride solution, thus, we achieve the control of cellproliferation using our porous film, which is important for cell investigation, searchfor the genes which can control the cell size and shape, and the investigation ofdifferent kinds of diseases. In addition, if we assemble the negatively charged DNAand positively charged PEI on the inner surface of the pores before cell adhesion, wecan achieve the in-situ cell transformation in the pores. Moreover, the transformationefficiency increases with more deposition cycle.
In a word, we develop a microemulsion method to achieve the fabrication of ordered honeycomb-patterned film. We can realize the convenient preparation of thewater soluble material pattern, such as protein pattern through this microemulsionmethod. In addition, through modifying the inner surface of the ordered pores, we canachieve the adhesion of cells in the pores, thus form ordered cell pattern. Cellinvestigations in the pores can also be taken based on this cell pattern. We believe thatthe results we got are very important for cell investigation, and the investigation andcontrol of many kinds of diseases.
本论文中，以水蒸气模板法为借鉴，以简化生命活性物质图案化结构制备过程为目的，发展了一种新颖的制备有序多孔结构的方法，利用这种方法，我们在制备得到有序蜂窝状多孔膜的同时获得了生命活性物质的图案化结构，而且也对此体系中的生命活性物质进行了进一步的研究。论文的研究内容主要分为以下三个部分：1、发展一种以微乳液为成膜体系，微乳液中的水滴为模板制备有序蜂窝状多孔膜的普适方法–微乳液滴模板法，并且研究此方法的各种影响因素，基于对各种影响因素的分析和讨论，提出此方法制备有序多孔膜的机理；2、由于在微乳液滴模板法中，作为模板的水滴来自于成膜体系而非从外界引入，所以通过将水溶性的蛋白质事先溶解在水相，可将蛋白质引入到制得多孔膜的孔洞内部，进而制备得到与孔洞排列相近的蛋白阵列，实现蛋白图案化结构的一步制备；3、以纯水作为水相，可制备孔洞内壁负载有正电荷表面活性剂的有序多孔结构，以此表面活性剂层为基础，可以组装与细胞带相反电荷的聚电解质，进而实现细胞在孔洞内部的吸附，由于选用细胞的大小与孔洞尺寸相近，所以可制备得到每个孔洞内只有一个细胞存在的单细胞阵列，进一步的，可研究细胞在孔洞内的行为，当组装的聚电解质为DNA时，还可实现细胞在孔洞内部的原位转染，进而研究组装层数对转染效率的影响。
以上研究工作为有序多孔结构的制备提供了一种新的方法，同时简化了水溶性生物材料的图案化结构制备过程，此外，以上研究工作的结果对细胞行为的研究也具有重要的应用价值。
Micropattern of biomaterials has attracted considerable attention due to their usein tissue engineering, cell investigation and biological detection. Various methodshave been developed for the fabrication of biomaterial patterns, top-down techniquesare the widely used ones. These methods are feasible, but the expensive equipmentand complex operation steps make them inconvenient for their application in manyareas. Self-assembly methods are more convenient compared to the top-downtechniques. Recently, breath figure method is attractive due to the use of cheap andnontoxic water droplets as template. However, the introduction of functional materialsalways realized by doping in organic solution or through complex introduction steps,which limit the functionalization with water soluble functional materials especiallywith brittle water soluble materials such as protein. Therefore, it is important todevelop a more simple method to achieve the fabrication of biomaterial pattern.
In this dissertation, we develop a microemlsion method which also uses thecheap and nontoxic water droplets as template. We realize the convenient fabricationof biomaterial pattern through this microemulsion method. Using thehoneycomb-patterned porous film prepared by this microemulsion method as atemplate, ordered cell pattern can be obtained. Furthermore, the activities of cells onthe honeycomb-patterned porous film can be investigated.
First, to simplify the introduction process of the water soluble materials, wedevelop a microemulsion method which also uses water droplets as template for thefabrication of ordered porous film. We use the microemulsion as casting solution,water droplets which come from the water phase of the microemulsion as template forthe honeycomb-patterned porous structure in this microemulsion method. Through thesize comparison of primary water droplets in micoemulsion and the pores in porousfilm, we found that the pores are much larger than the primary water droplets, thus thepores were formed by the fusion of primary water droplets in microemulsion.Furthermore, we also investigate the influence of solvent, polymer, surfactant, volumeratio of water phase and casting volume on the formation and order of the porous film.The surfactant distribute at oil-water interface to stabilize the water droplets due totheir amphiphilicity, thus the addition of surfactant is critical for the formation ofordered porous structure. The order and pore size of the porous film can be adjustedby varying the concentration of surfactant. In addition, solvent, polymer, volume ratioof water and casting volume can also influence the order and pore size of the porousfilm through affecting the evaporation speed of solvent and the stability of the waterdroplets. The investigations of these factors also confirm the existence of water fusionduring the formation of porous film. We propose a possible mechanism for thefabrication of ordered honeycomb-patterned porous film through microemulsionmethod based on the analysis of all the results, which is valuable for thefunctionalization of the porous film.
Second, in the microemulsion method, the water droplets come from water phaseof the microemulsion, therefore, if we add the water soluble functional material intowater phase beforehand, it will be exist in the water droplets during the formation ofordered porous film and then fall down to the bottom of the pores with theevaporation of water, thus form the ordered porous film which was functionalized bythis water soluble material. Based on the above analysis, we dissolved the protein intowater phase aforehand, and then use this protein aqueous solution as a water phase toprepare the protein contained microemulsion. After casting this protein contained microemulsion onto proper substrate, protein contained porous film can be obtained.Through the confocal laser scanning microscopy characterization after proteinlabeling with fluorescent dye, we found that most of the protein locate at the bottomof the pores, thus form the similar pattern with the pores. Therefore, we can preparethe ordered porous film and protein pattern at the same time. Furthermore, thepatterned protein can interact with the molecular that can specifically recognize it,thus we can distinguish different kinds of protein using this protein pattern.
Third, during the formation of ordered porous film, the surfactant locate at theoil-water interface, so it will be exist at the inner surface of the pores after the filmformation. In our system, we choose the positively charged DDAB as a surfactant,therefore, we can use this DDAB layer as a substrate for polyelectrolyte assemblybased on electrostatic interaction. After the assembly of positively chargedpolyelectrolyte on the inner surface of the pores, we can use this polyelectrolyte layeras a substrate to achieve the adhesion of negatively charged cell, and thus form thecell pattern similar to the array of the ordered pores. Here, we select the yeast cellswhich have the similar size to the patterned pores. Therefore, we can prepare orderedcell pattern with single cell in one pore, which is important for the investigation ofsingle cell. And the viability of the yeast cells retained through the FDA test.Furthermore, the yeast cells can grow larger till form the similar morphology with thepores after culturing for12hours, but their proliferation was inhibited due to the limitof the pores. The grown cells can proliferate again after releasing from the poresthrough immersing into sodium chloride solution, thus, we achieve the control of cellproliferation using our porous film, which is important for cell investigation, searchfor the genes which can control the cell size and shape, and the investigation ofdifferent kinds of diseases. In addition, if we assemble the negatively charged DNAand positively charged PEI on the inner surface of the pores before cell adhesion, wecan achieve the in-situ cell transformation in the pores. Moreover, the transformationefficiency increases with more deposition cycle.
In a word, we develop a microemulsion method to achieve the fabrication of ordered honeycomb-patterned film. We can realize the convenient preparation of thewater soluble material pattern, such as protein pattern through this microemulsionmethod. In addition, through modifying the inner surface of the ordered pores, we canachieve the adhesion of cells in the pores, thus form ordered cell pattern. Cellinvestigations in the pores can also be taken based on this cell pattern. We believe thatthe results we got are very important for cell investigation, and the investigation andcontrol of many kinds of diseases.
引文
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