Synthesis of phosphonate functionalized silica nanoparticles for protein immobilization, intracellular protein delivery and catalytic applications
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Organosilica nanoparticles have attracted a lot of research interest in a variety of areas such as drug delivery and catalysis because of their properties which include high surface area as well as tunable particle and pore size. In particular, nanoparticles with large pore sizes are of great interest because of their potential to host large guest molecules such as proteins and as catalysts. The focus of the work in the thesis was to develop phosphonate functionalized organosilica nanoparticles for biomedical and catalytic applications. Raspberry textured phosphonatesilica nanoparticles denoted, RNPPME(2.5) (where the number in the brackets represents the moles of organophosphonate per gram), with large pore size (11–17 nm), uniform particle size (70 – 90 nm) and high surface area were produced through the use of template directed base catalysed synthesis, using tetraethylorthosilicate (TEOS) and dimethylphosphonatoethyltrimethoxysilane (DMPTMS) as the silica sources. The role of the reaction conditions such as temperature, surfactant concentration, pH, organosilane concentration and type were investigated and a mechanism for the raspberry nanoparticle formation was proposed. The particles were characterized using electron microscopy (SEM and TEM), Dynamic light scattering (DLS), silicon and phosphorus solid state NMR, and solution phase proton NMR of base digested particles, FT–IR, nitrogen adsorption porosimetry and thermal analysis (TGA). The ability of the particles to host protein molecules of the model protein, bovine serum albumin (BSA) was investigated and the particle–protein composite was characterized using circular dichroism (CD). Raspberry textured nanoparticles were found to host large quantities (26 wt%) of protein. Studies on other (small pore (3 nm) phosphonate functionalized nanoparticles NP_PME(0.2) and NP_PME(1.0)) and (3 nm pore) unfunctionalized mesoporous silica nanoparticles (MSN) revealed that phosphonate loading and the pore size influenced the protein uptake In addition to high protein uptake, the RNP_PME(2.5) particles also absorbed protein molecules rapidly (~ 20 minutes to maximum load). CD studies determined that the particle bound protein structure was not affected at physiological pH (7.40). The vast majority of the previously reported studies involving protein immobilization involved the use of bulk silica materials, which cannot be dispersed and hence those materials were unsuitable for in vivo protein delivery applications. The BSA@RNP_PME(2.5) particles showed good protein load and dispersion properties and hence are excellent protein delivery agents. Dispersions of nanoparticle composite BSA#@RNP*_PME(2.5) (where BSA# represents fluorescein isothiocyanate labelled BSA and RNP*_PME(2.5) represents rhodamine B isothiocyanate labelled RNP_PME(2.5)) was used to successfully deliver membrane impermeable protein BSA into HeLa cells. Intracellular protein delivery has attracted great interest due to its potential therapeutic applications and research tool value (e.g. for studying various cellular pathways). The toxicity of the guest free particles RNP*_PME(2.5) and the protein loaded particles BSA#@RNP*_PME(2.5) was studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The particles and the protein-particle composite were found to be non-toxic. The mechanism of the particle uptake by the cells was also studied. The unloaded (protein free) particles were found to be taken up by caveolar endocytosis pathway and the protein loaded particles were taken up by folic acid mediated pathway. The results indicated that the particles can successfully deliver membrane impermeable protein across the cell membrane. This result suggested that the particles can potentially be used for intracellular protein delivery applications. Raspberry textured nanoparticles RNP_PME(2.5) were also used to host the enzyme lipase. It was demonstrated that immobilization increased the maximum velocity and Michaelis constant of the enzyme and also that the particles offered protection against the denaturing agent, urea. Finally, in a chemical catalysis application, the RNP_PME(2.5) particles were used to synthesize the platform chemical HMF, through Brönsted acid catalysed dehydration of fructose. High yields of HMF (87 %) were achieved when 10 wt% fructose was used. The particles demonstrated good recyclability and also the ability to convert up to 50 wt% fructose into HMF (80 % yield). The particles therefore acted as protective agents for enzymes and can therefore be used as enzyme immobilizing agents. Additionally, they also acted as excellent Brönsted acid catalysts.
AuthorsMaddala, Sai P.
- Theses