UVM Theses and Dissertations
Format:
Print
Author:
Cheng, Kai
Title:
Dept./Program:
Chemistry
Year:
2008
Degree:
PhD
Abstract:
The recent endeavors on the development of synthetic porous materials, especially mesoporous silica materials, for their great potentials in practical applications such as bioadsorption, separation, biocatalysis, biosensors, and vehicles for drug delivery, are based on their very attractive features: thermally and mechanically stable mesoporous structure, narrow and tunable pore size distribution, extremely high specific surface area and pore volume, non-toxic nature, good biocompatibility. A monodisperse, spherical mesoporous silica (Acid-Prepared Mesoporous Spheres, APMS) was prepared and then functionalized with two types of Fmoc (9-fluorenylmethyloxycarbonyl) terminated silanes with variable chain lengths. N₂ physisorption experiments indicated that, under some conditions, the pores of the solid were completely filled by the Fmoc-protected organosilanes. The rate of deprotection was followed by UVIvisible spectroscopy, and a plot of Fmoc released versus time showed a sigmoidal shape. The location of the deprotection reaction in the pores of the silica can be empirically controlled. Our work provides a method by which the surface of the porous silica can be hnctionalized in a well-defined manner.
The diffusion-reaction issues in the thiol modified mesoporous silica spheres obtained by either calcination/graft or graft/extraction route have been studied in detail from the quantitative activation of the grafted thiol groups and the optimization of the immobilization of the CD4 fragments on the silica. The accessibility to the binding sites and the rate of the thioYdisulfide interchange reaction within the silica are dependent on the porosities of the silica samples and the distribution of the finctional groups within the pores. The mesoporous silica spheres with free amine groups exposed solely on external surfaces were prepared by a protection/deprotection strategy and then derivatized with the crosslinkers to introduce surface thiol groups. The thermodynamic and kinetic studies of the immobilization of cysteine-incorporated CD4 fragments on the silica spheres have highlighted the optimal conditions by which the peptides can be covalently anchored to the external surface of a mesoporous silica spheres. The covalent attachment of three heterobifunctional crosslinkers on the thiolated silica spheres through either the disulfide bonds or stable thioether linkages while retaining NHS-ester activity for subsequent reaction with primary amine on the proteins or the hydrazide activity for subsequent reaction with oxidized carbohydrate residues in glycoprotein (CD4) were confirmed by UV-vis spectrophotometric method and FTIR spectroscopy. Although the immobilization of CD4 on the solid particles via amino groups significantly reduced its gp120 binding affinity, the immobilization via the oxidized carbohydrate residues enabled the CD4 protein attaching to the solid surface in homogeneous manner without loss of any gp120 binding affinity.
A series of functionalized APMS (functionalized with thiol, primary amine, and secondary amine moieties) were prepared by either calcination/grafting or grafting/extraction route. Subsequently, the octadecene and tetraethylene glycol (TEG) moiety were covalently immobilized on the surface of functionalized APMS via either amine bond, disulfide bond, or amide bond. APMS-TEG showed enhanced uptake by both lung epithelial and malignant mesothelioma (MM) cells in vitro (less than 2 hours) compared to APMS with different coatings. APMS-TEG can be used as a vehicle to deliver constructs or anti-cancer drugs into the cell and freely interact with intracellular target molecules for enhanced effects. The adsorption and release of doxorubicin (DOX) within/from various functionalized APMS samples has increased DOX cytotoxicity in human MM cells in vitro. Compared with DOX alone ranging from 5-2000 nM, APMS-TEG as vehicles to carry DOX into cells even at a range of low concentrations from 50 to 80 nM can lead to a dramatic increase in cell toxicity.
To functionalize the external surface with two types of organic functional moieties in a well-defined, controllable manner, we successfully developed a Stöber nanosphere assisted surface-modification method by which the distribution and positions of the functional sties for subsequent protein coupling on the silica surface were very well controlled. The resulting bifunctional silica spheres were covalently immobilized with both the tetraethylene glycol (TEG) moieties and anti-human mesothelioma antibody. The ratio of the amount of antibody to TEG moiety was optimized to enhance the efficiency of uptake of modified silica particles by MM cells by adjusting the size of Stöber spheres and the density of the array of Stober spheres during the Stöber-assisted surface modification. Enhanced efficiency of uptake by mesothelioma cells and improved selectivity of targeting delivery were observed in a co-culture study with human mesothelioma and lung cancer by using confocal microscopty and flow cytometry.
The diffusion-reaction issues in the thiol modified mesoporous silica spheres obtained by either calcination/graft or graft/extraction route have been studied in detail from the quantitative activation of the grafted thiol groups and the optimization of the immobilization of the CD4 fragments on the silica. The accessibility to the binding sites and the rate of the thioYdisulfide interchange reaction within the silica are dependent on the porosities of the silica samples and the distribution of the finctional groups within the pores. The mesoporous silica spheres with free amine groups exposed solely on external surfaces were prepared by a protection/deprotection strategy and then derivatized with the crosslinkers to introduce surface thiol groups. The thermodynamic and kinetic studies of the immobilization of cysteine-incorporated CD4 fragments on the silica spheres have highlighted the optimal conditions by which the peptides can be covalently anchored to the external surface of a mesoporous silica spheres. The covalent attachment of three heterobifunctional crosslinkers on the thiolated silica spheres through either the disulfide bonds or stable thioether linkages while retaining NHS-ester activity for subsequent reaction with primary amine on the proteins or the hydrazide activity for subsequent reaction with oxidized carbohydrate residues in glycoprotein (CD4) were confirmed by UV-vis spectrophotometric method and FTIR spectroscopy. Although the immobilization of CD4 on the solid particles via amino groups significantly reduced its gp120 binding affinity, the immobilization via the oxidized carbohydrate residues enabled the CD4 protein attaching to the solid surface in homogeneous manner without loss of any gp120 binding affinity.
A series of functionalized APMS (functionalized with thiol, primary amine, and secondary amine moieties) were prepared by either calcination/grafting or grafting/extraction route. Subsequently, the octadecene and tetraethylene glycol (TEG) moiety were covalently immobilized on the surface of functionalized APMS via either amine bond, disulfide bond, or amide bond. APMS-TEG showed enhanced uptake by both lung epithelial and malignant mesothelioma (MM) cells in vitro (less than 2 hours) compared to APMS with different coatings. APMS-TEG can be used as a vehicle to deliver constructs or anti-cancer drugs into the cell and freely interact with intracellular target molecules for enhanced effects. The adsorption and release of doxorubicin (DOX) within/from various functionalized APMS samples has increased DOX cytotoxicity in human MM cells in vitro. Compared with DOX alone ranging from 5-2000 nM, APMS-TEG as vehicles to carry DOX into cells even at a range of low concentrations from 50 to 80 nM can lead to a dramatic increase in cell toxicity.
To functionalize the external surface with two types of organic functional moieties in a well-defined, controllable manner, we successfully developed a Stöber nanosphere assisted surface-modification method by which the distribution and positions of the functional sties for subsequent protein coupling on the silica surface were very well controlled. The resulting bifunctional silica spheres were covalently immobilized with both the tetraethylene glycol (TEG) moieties and anti-human mesothelioma antibody. The ratio of the amount of antibody to TEG moiety was optimized to enhance the efficiency of uptake of modified silica particles by MM cells by adjusting the size of Stöber spheres and the density of the array of Stober spheres during the Stöber-assisted surface modification. Enhanced efficiency of uptake by mesothelioma cells and improved selectivity of targeting delivery were observed in a co-culture study with human mesothelioma and lung cancer by using confocal microscopty and flow cytometry.