Cationic Ligands Functionalized Gold Nanoparticles For Intracellular Delivery And Microcapsule Self-assembly Applications

Gold nanoparticles (AuNPs) are versatile nanomaterials with unique physical and chemical properties. The typical physical properties of AuNPs include surface plasmon resonance, enhancement of magnetic resonance imaging, surface-enhanced Raman scattering, and enhancement and quenching of fluorescence. The chemical properties of AuNPs are represented by their oxidation resistance, bio–inertness, low cytotoxicity, and readily conjugate with thiolated compounds through Au–S bond. Moreover, the modification of AuNPs with functional thiolated ligands could combine the essential physical and chemical properties of the AuNPs and the chemical properties of the ligands, which impart the AuNPs new chemical and biological properties with applications as diverse as delivery, sensing, imaging and therapies. In this thesis, we designed and synthesized several thiolated cationic ligands. These functional ligands were used for AuNPs surface modification by using place–exchange strategy resulting in positively charged functional AuNPs, and the applications of the functionalized AuNPs for intracellular delivery and microcapsule self–assembly were studied.Cationic peptide ligand functionalized AuNPs as carrier for gene delivery. Cationic peptides RRR, KKK, KRK and HKRK were conjugated to thiol compound and used for AuNPs surface modification resulting in water insoluble RRR–AuNPs, and water soluble KKK–AuNP, KRK–AuNPs and HKRK–AuNPs. Based on the excellent water solubility, KKK–AuNP, KRK–AuNPs and HKRK–AuNPs were used as gene delivery carrier for the transfection of 293T cells withβ–gal reporter gene plasmid. The results showed that all of the three nanoparticles efficiently transfected 293T cells as determined byβ–gal activity. The transfection efficiency of the three nanoparticles were determined as HKRK–AuNPs>KRK–AuNPs>KKK–AuNPs. Maximum transfection was observed by using HKRK–AuNPs as carrier. The cell viability assay indicated that KKK–AuNPs were moderately toxic. However, full retention of cell vitality was observed for both KRK–AuNPs and HKRK–AuNPs. The TEM images revealed that the cell uptake mechanism for AuNPs was endocytosis.Cationic peptide ligand functionalized AuNPs as carrier for enzyme delivery. According to the gene delivery result, HKRK–AuNPs was a highly effective and low toxic carrier. To further explore the delivery properties of HKRK–AuNPs, the nanoparticle was used as enzyme carrier to transferβ–gal into HeLa, COS–1, C2C12 and MCF7 cells. The results indicated that theβ–gal conjugated to HKRK–AuNPs through electrostatic interaction forming HKRK–AuNPs/β–gal. Circular dichroism (CD) spectra revealed that HKRK–AuNPs had minimal effect on the secondary structure ofβ–gal. HKRK–AuNPs were able to transportβ–gal into all the four cell lines tested in the study. The temperature dependent experiment showed that the enzyme internalization via endocytosis. Most importantly, the transported enzymes were able to escape from endosomes and the enzymatic activity was kept after the delivery. Cell viability evaluated by alamar blue assay, trypan blue exclusion assay and calcein AM assay demonstrated no apparent cytotoxicity of the HKRK–AuNPs, which implied the nanoparticle an excellent carrier.Cationic quaternary ammonium (TTMA) ligand functionalized AuNPs for catalytic microcapsule self–assembly. The immobilization of enzymes on a template with high surface to volume ratio would increase the immobilization efficiency and the utility efficiency of the enzyme. For this purpose, O/W droplet provide and ideal geometry for enzyme immobilization. However, the lack of strong binding force between the droplet and enzyme limited the confinement of enzyme at the O/W droplet interface. To investigate the mimmobilization of enzymes on the O/W droplet surface, AuNPs were functionalized with cationic TTMA ligands resulting in positively charged TTMA–AuNPs. These cationic nanoparticles were conjugated toβ–gal forming TTMA–AuNPs/β–gal conjugates which were used for microcapsules self–assembly at O/W droplet interface. The results indicated that no self–assembly phenomenon was observed for both free TTMA–AuNPs and freeβ–gal. However, the TTMA–AuNPs/β–gal conjugates could be self–assembled onto the O/W droplet surface resulting in stable catalytic microcapsules. The surface charge density difference of the TTMA–AuNPs,β–gal and TTMA–AuNPs/β–gal conjugates revealed that the driven force for this self–assembly process is surface charge density decrease. Significantly, 99% of the freeβ–gal molecules were immobilized onto the O/W droplet surface and 76% of the enzymatic activity preservation was observed after the immobilization.Cationic arginine (Arg) ligand functionalized AuNPs for self–assembly of drug delivery nanocapsules. Nanoparticles stabilized microcapsules are of great potential in drug delivery applications. However, current methods can only generate microcapsules with size ranging from 1~50μm, limiting their effectiveness as delivery vehicles. The self–assembly of nano–sized microcapsules (nanocapsules) is still a chanllenge due to the presence of interfacial energy and Laplace pressure at the O/W droplets interface. To address this issue, AuNPs were functionalized with arginine liagnds, and linoleic acid was emulsified to generate nano–sized O/W droplet template. The strong hydrogen bonds between the arginine group on the Arg–AuNPs surface and carboxyl group at the terminal of the linoleic acid molecules guided the self–assembly of the nanoparticles at the O/W droplet interface forming stable nanocapsules. To further stabilize the nanocapsule, the positively charged Arg–AuNPs on the nanocapsule surface were crosslinked by negatively charged transferrin. The size of the nanocapsules was ~100 nm determined by TEM and DLS analysis. The application of the prepared nanocapsules for anti–cancer drug, paclitaxel, delivery was studied with HeLa cells. The nanocapsules showed no measurable toxicity, whereas efficient delivery and high cytotoxicity was observed with paclitaxel loaded nanocapsules. The prepared nanocapsule is supposed to be an excellent drug delivery vehicle.