【作者】 何定庚；

【导师】 何晓晓； 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2012， 博士

【摘要】 介孔二氧化硅由于具有超高的比表面积、大的介孔体积、均一可调的孔径尺寸和有序的介孔结构等独特的介观结构和物理化学性质，在催化、吸附、大分子转化、蛋白质分离鉴定和光电磁材料等高科技领域，尤其是在刺激响应可控释放领域以及分析检测方面拥有广阔的应用前景。但是，目前已发展的许多刺激响应控制释放系统和分析检测系统均缺乏可逆性和可再生性。基于此，本论文以介孔二氧化硅纳米颗粒为载体材料，结合胸腺嘧啶的光反应性质和DNA分子构型的多样性，构建了基于光、pH、生物分子响应的可逆控制释放系统，同时结合磁纳米颗粒的磁分离特性，设计了一种能同时检测和移除汞离子的可再生核壳磁介孔二氧化硅纳米颗粒。主要开展以下研究工作：一、基于胸腺嘧啶功能化介孔二氧化硅的光响应可逆控制释放系统胸腺嘧啶具有良好的可逆光反应性质，是一种理想的光激发门控分子。本章将胸腺嘧啶共价交联到介孔二氧化硅纳米颗粒表面，设计了一种光响应可逆控制释放系统。在波长为365nm紫外光照射下，介孔二氧化硅颗粒表面的胸腺嘧啶形成二聚体结构，封堵住介孔，阻止包裹客体分子的释放；在波长为240nm的紫外光照射下，胸腺嘧啶二聚体发生解离，使封堵的介孔打开，释放装载的客体分子。Ru（bipy）32+分子由于其良好的光学性质而被选作模式客体分子，研究了其在不同波长光照条件下的储存和释放情况。此外，利用Ru（bipy）32+分子荧光的氧淬灭性质，我们成功设计了一种可再生的光开关氧传感器。二、基于i-motif DNA和羟基孔雀石绿功能化介孔二氧化硅的光响应可逆控制释放系统寡核苷酸因其具有良好的生物相容性、构型的多样性和稳定的物理化学性质等而成为构建“纳米门”的理想材料。本章选择i-motif DNA（富含胞嘧啶的四链DNA）作为pH响应的“纳米门”，羟基孔雀石绿（MGCB）作为光激发氢氧根离子发射器，结合介孔二氧化硅纳米颗粒巧妙地设计了一种新型的光响应可逆控制释放系统。首先，在介孔颗粒表面共价修饰上i-motif DNA，然后将MGCB通过静电吸附和疏水作用力固定在介孔通道壁上，并选择Ru（Bipy）32+作为模式客体分子装载进介孔通道内。当溶液pH为5.0时，i-motif DNA能有效地封堵介孔，限制客体分子的释放。在紫外光照射条件下，MGCB解离出氢氧根离子，导致溶液pH值升高，使得i-motif DNA去折叠成单链结构，从而导致介孔的打开和客体分子的释放。在黑暗的条件下，通过再结合溶液中的氢氧根离子，MGCB分子得以再生，使溶液的pH值返回到原始值，此时单链DNA再次折叠成i-motif DNA结构，从而关闭介孔，完成一个“开/关”循环。因此，通过交替地打开和关闭光源，DNA构型的变化和介孔的打开与关闭能被反复操作，达到可逆循环的效果。该方法利用光刺激pH变化分子来间接诱导pH敏感DNA的构型变化，简便易行，并且不需要复杂的合成技术，为纳米可控释放技术在实际体系中的应用奠定了基础。三、基于T-Hg2+-T碱基对介导的双链DNA功能化介孔二氧化硅的细胞内pH响应可逆控制释放系统胸腺嘧啶（T）能特异性结合汞离子形成T-Hg2+-T结构。在中性条件下，T-Hg2+-T结构具有比A-T结构更高的稳定性，而在弱酸性条件下能发生解离。本章将T-Hg2+-T碱基对介导的双链DNA共价交联在介孔二氧化硅表面，发展了一种细胞内酸响应可逆控制释放系统。在中性条件下，双链DNA能有效封堵介孔，防止药物分子的泄漏；而在弱酸（pH5.0）条件下，由于T-Hg2+-T结构的解离，双链DNA解链成单链，介孔被打开，装载的药物分子被释放。阿霉素（Dox）是一种常见的化疗药物分子，因此将其包裹在介孔通道内，以T-Hg2+-T碱基对介导的双链DNA封堵介孔，考察该系统在细胞内的控制释放行为。结果表明，细胞内溶酶体pH值能刺激介孔打开，释放Dox分子。此外，MTT实验结果表明，该药物释放系统展现良好的生物相容性，是一种理想的细胞内酸响应药物载体，有望应用于活体内酸响应药物释放研究。四、基于C-Ag+-C碱基对介导的双链DNA功能化介孔二氧化硅的生物分子响应可逆控制释放系统细胞在生长代谢过程中会产生一系列特殊功能的生物分子，因此发展一种生物分子响应的控制释放系统对药物运输和肿瘤靶向治疗具有重大意义。本章利用C-Ag+-C结构介导的双链DNA作为分子门，结合介孔二氧化硅纳米颗粒设计了一种生物分子响应可逆控制释放系统。在这个系统中，富含胞嘧啶的DNA（C-richDNA）被共价交联在介孔二氧化硅颗粒表面。当银离子存在时，邻近的C-rich DNA能相互结合，形成具有C-Ag+-C结构的双链DNA，从而封堵住介孔，阻止客体分子的释放。巯基类生物分子（GSH和半胱氨酸等）能特异性螯合C-Ag+-C结构中的银离子，使得该双链DNA变性解离，进而打开介孔，释放客体分子。我们利用Ru（bipy）32+作为模式客体分子，将其包裹进介孔通道内；以二硫苏糖醇（DTT）作为模式刺激分子，考察该系统的刺激响应释放行为。实验结果表明，在DTT存在条件下，Ru（bipy）32+能很好地实现控制释放。此外，该DNA分子的开关态能通过银离子和DTT的交替加入来进行调节。同时，该系统能通过胞吞方式进入细胞，并且展现出非常低的细胞毒性（IC50>200μg mL-1）。这些特性使得该DNA分子门控释放系统有望用于细胞内的药物控制释放。五、可再生多功能磁介孔二氧化硅颗粒用于汞离子检测和移除汞离子是水环境中的一种重金属污染物，毒性高，损害人类健康。本章结合磁性纳米颗粒的磁富集特性和介孔二氧化硅纳米颗粒的优点，设计了一种核壳结构的磁介孔二氧化硅纳米颗粒，然后通过EDC/NHS策略分别将能与汞离子特异性结合的DNA（T-rich DNA）和胸腺嘧啶（T）修饰在介孔颗粒表面和介孔通道内部，构建了一种能同时检测和移除汞离子的纳米传感器（Fe3O4@nSiO2@mSiO₂-T-TRDNA）。该传感器利用单链T-rich DNA捕获溶液中的汞离子而形成双链结构，以SYBR Green I（特异性嵌入双链而使得荧光增强的染料）对该双链DNA进行染色来达到检测汞离子的目的；而汞离子的移除主要是通过介孔通道内固定的大量能与汞离子特异性结合的胸腺嘧啶来实现。实验结果表明，该纳米传感器不仅实现了理想条件下汞离子检测和移除（检测限为2nM），而且能对环境污染水样中汞离子进行检测和快速移除。此外，该传感器在简单的酸处理下能再生，并且固定在颗粒表面的DNA具有很好的抗酶切稳定性。因此，该可再生多功能介孔二氧化硅颗粒能用于环境水样中汞离子的特异性检测和选择性移除。更多还原

【Abstract】 Mesoporous silica nanoparticles, due to its high surface area, large mesoporousvolume, uniform tunable size （2-30nm）, ordered mesoporous structure （multiplearrangement）, the unique mesoscopic structure and physical and chemical properties,show a broad application in catalysis, adsorption, macromolecules transformation,protein isolation, optical electromagnetic materials, especially in stimuli-responsivecontrolled release and biosensors. But recent controlled release systems have somedisadvantages such as lack of reversibility and reproducibility. Consequently, thispaper will employ mesoporous silica nanoparticles as the nanocarriers to design thereversible stimuli-responsive release system and develop reproducible methods formercury detection and removal. The main research aspects are as follow:1. A light-responsive reversible controlled release system using thymine-modifiedmesoporous silica nanoparticlesIn this paper, a reversible light-responsive controlled release system based onmesoporous silica nanoparticles （MSN） functionalized with thymine derivatives isdesigned and demonstrated. The closing/opening protocol and release of the entrappedguest molecules is related by a photodimerization-cleavage cycle of thymine upondifferent irradiation. In the system, thymine derivatives with hydrophilicity andbiocompatibility were grafted on the pore outlets of MSN. The irradiation with365nm wavelength of UV light to thymine-functionalized MSN leaded to the formation ofcyclobutane dimer in the pore outlet, subsequently resulted in blockage of pores andstrongly inhibited the diffusion of guest molecules from pores. With240nmwavelength of UV light irradiation, the photocleavage of cyclobutane dimer openedthe pore and allowed release of the entrapped guest molecules. As aproof-of-the-concept, Ru（bipy）32+was selected as the guest molecule. Then thelight-responsive loading and release of Ru（bipy）32+have been investigated. Theresults indicated that the system had excellent loading amount （53μmol g-1MSN） andcontrolled release behavior （82%release after irradiation for24h）, and thelight-responsive loading and release procedure exhibited a good reversibility. Besides,the light-responsive system loaded with Ru（bipy）32+molecule could also be used as alight-switchable oxygen sensor.2. A photoresponsive reversible controlled release system using i-motif DNA andMGCB functionalized mesoporous silica nanoparticles This paper proposed a novel photoresponsive reversible controlled release systemusing mesoporous silica nanoparticles functionalized with i-motif DNA and malachitegreen carbinol base （MGCB）. In this system, MGCB was immobilized on thenanochannel walls of MSN as a light-induced hydroxide ion emitter, and i-motif DNAwas grafted on the surface of MSN as a cap. The photoirradiation with365nmwavelength of UV light made MGCB dissociate into malachite green （MG） cation andOH–ion, which induced i-motif DNA to unfold into the single-stranded form. Thus,the pores were uncapped and the entrapped guest molecules were released. After thelight was turned off, the MG cation recombined with the OH–ion. The single-strandedDNA switched back to i-motif structure to cap the pore again. Rooting form MGCBmediated DNA conforma-tion changes, the quadruplex DNA-gated switch could beeasily operated by turning the light on or off. Importantly, the opening/closingprotocol was highly reversible and a partial cargo release could be easily achieved atwill. This proof of concept might promote the application of DNA in the controlledrelease and could also provide an idea to design various photon-fueledcontrolled-release systems by using a combination of photoirradiated pH-jump systemand other kinds of pH-sensitive linkers.3. A reversible intracellular acid-responsive controlled release system using T-Hg2+-Tbase pairs mediated double-stranded DNA-capped mesoporous silica nanoparticlesThis paper proposed a reversible intracellular pH-responsive controlled releasesystem consisting of mesoporous silica nanoparticles （MSN） functionalized on thepore outlets with T-Hg2+-T base pairs mediated double-stranded DNA （dsDNA1）. Inthis system, the dsDNA was grafted on the MSN surface as a nanoscopic cap. Thecontrolled release system was closed at neutral pH but opened at pH5.0due to thedissociation of T-Hg2+-T structures and the subsequent melting of dsDNA1. As aproof-of-the-concept, the doxorubicin （Dox） was loaded into the dsDNA1-modifiedMSN （MSN-dsDNA1） as a model drug. Release-profile studies in water showed thatno Dox leaked when the cap was closed and that release occurred immediately afteracidification. By alternately changing the pH from5.0to7.2, the DNA cap could beswitched “on” and “off” and thereby regulated the partial release of Dox. Further invitro studies demonstrated that the Dox-loaded MSN-dsDNA1（MSN-Dox-dsDNA1）could be endocytosed and accumulated within endosomes and lysosomes, followed byserving as a delivery for the controlled release of Dox into the cell nuclei at theendosomal and lysosomal pH level inside live HeLa cells. The cell viability resultsshowed that the inhibitory concentration （IC50） of MSN-Dox-dsDNA1was low （≈ 12.5μg mL–1）, while MSN-dsDNA1（IC50>100μg mL–1） had a negligiblecytotoxicity at the same concentration, indicating that MSN-dsDNA1was fairlybiocompatible and indeed served as a drug-carrier for intracellular controlled release.We believe that the nanosystem may prove to be a significant step toward thedevelopment of an intracellular acid-responsive drug delivery system that is apromising candidate in vivo delivery of therapeutic agents.4. Reversible bioresponsive controlled release system using mesoporous silicananoparticles capped with C-Ag+-C base pairs mediated double-stranded DNAWe reported a novel reversible bioresponsive controlled-release system consistingof mesoporous silica nanoparticles （MSN） functionalized with C-Ag+-C base pairsmediated double-stranded DNA. In this system, a unique sequential cytosine （C）-richDNA as the smart molecule-gated switch was grafted on the mesoporous silicananoparticles （MSN） surface. In the presence of Ag+ions, the closer C-rich DNAcould hybridize each other by the formation of C-Ag+-C structure based onmetal-dependent pairs of two nucleobases, resulting in blockage of pores and packageof guest molecules. By a competitive displacement reaction, the duplex DNA withC-Ag+-C structure deformed into single-stranded DNA in the presence ofthiol-containing molecule, such as dithiothreitol （DTT）, which gave rise to uncappingand the subsequent release of the entrapped guest molecules. The reversible open andclosed states of DNA molecule-gated switch could be easily achieved by alternatingaddition of Ag+-linkers and DTT molecules. Our results demonstrated that the systemhad excellent loading amount （43μmol g–1） and good controlled release behavior.Moreover, the system could enter the cells through endocytosis and showed a lowcytotoxicity even with treatment in a high concentration （200μg mL–1）. We believedthat the stimuli-responsive controlled MSN release system based on the smartmolecule-gated switch could play an important role in the development intracellulardelivery nanodevices.5. Regenerable multifunctional mesoporous silica nanocomposites for simultaneousdetection and removal of mercury （II）Mercury (Hg²⁺) is a highly toxic and widespread environmental pollutant. Herein, aregenerable core-shell structured magnetic mesoporous silica nanocomposite withfunctionalization of thymine （T） and T-rich DNA （denoted as Fe3O4@nSiO2@mSiO2-DNA-thymine） has been developed for simultaneous detection and removal ofHg²⁺. The detection mechanism is based on Hg²⁺-mediated hairpin structure formedby T-rich DNA functionalized on the surface of the nanocomposite, where, upon addition of SYBR Green I dye, strong fluorescence is observed. In the absence ofHg²⁺, however, addition of the dye results in low fluorescence. The limit of detectionfor Hg²⁺in a buffer is2nM by fluorescence spectroscopy. Simultaneously, thenanocomposites-based sensors feature a selective binding with Hg²⁺between twothymines immobilized at the interior surface of the mesopores and exhibits efficientand convenient Hg²⁺removal by a magnet. Kinetic study reveals that the Hg²⁺removal is a rapid process. The applicability of the developed materials isdemonstrated to detect and remove Hg²⁺from samples of Xiangjiang river waterspiked with Hg²⁺. In addition, distinguishing aspects of the nanocomposite for Hg²⁺detection and removal also include the regeneration using a simple acid treatment andresistance to nuclease digestion. Similar process can be used to functionalizeFe3O4@nSiO2@mSiO2nanocomposite with other nucleic acids and small moleculesfor environmental and biomedical applications.更多还原