Over the last two decades, silica based nanoparticles (SiNPs) have been extensively investigated as promising nano-carriers to deliver various therapeutic/diagnostic agents into living systems, due to their unique properties of tunable pore structure and particle size, easy surface modification and low cost. In particular, SiNPs with small sizes ( 100 nm) and high monodispersity, SiNPs possess great advantages in cell endocytosis process, which is vital to achieve high efficiency in biomedical applications. Although there have been tremendous studies in the synthesis of monodisperse nanometer-sized SiNPs, more efforts are still needed to develop facile, economic and environmentally friendly synthesis approaches for fabricating novel monodisperse SiNPs with desired particle size, nano-structure and functionality. The as-designed novel SiNPs are expected to expand their capacity in various biomedical applications, such as enhanced bio-imaging performance in three dimensional spheroid models, improved cellular drug/gene delivery efficiency.The aim of this project is to develop novel and facile approaches to prepare highly monodispersed SiNPs with finely controlled structures for drug/gene delivery and gain insight into the roles of particle size, surface functionality on cell penetration performance and drug/gene delivery efficiency. The main achievements obtained in this thesis are listed below.In the first part, a new and facile approach has been developed to prepare monodisperse mesoporous silica nanospheres (MMSNs) with controlled particle sizes (50-100 nm) and pore diameters (2.8-4.0 nm). In this approach, MMSNs were synthesized simply in a sodium acetate solution without adding any other alkali or alcohol additives. By further investigations on formation process, we proposed a spherical micelle templating mechanism to explain the formation of MMSNs in our system, which is different from that of traditional highly ordered mesoporous silica nanoparticles (MCM-41). MMSNs developed in this part are expected to have potential applications in drug/gene delivery and cell imaging.In the second part, even smaller mono-dispersed SiNPs (ultra-small hybrid silica spheres, UHSS) with a diameter of only 10 nm were developed by a facile strategy under phosphate-citrate buffer solution (pH = 4.6) at room temperature without addition of toxic additives. Compared to traditional MCM-41 nanomaterials, the designed novel UHSS showed enhanced penetration ability in three dimensional glioma spheroids.Following the second part, epoxysilane functionalized UHSS (Epoxy-UHSS) with a diameter of 10 nm were designed under similar synthesis conditions, which can be easily covalently conjugated with cationic polyethyleneimine (PEI) (PEI-UHSS). This designed positively charged PEI-UHSS demonstrated excellent delivery efficiency of a functional siRNA against polo-like kinase 1 (PLK1-siRNA) in osteosarcoma cancer cells (KHOS) and survivin-siRNA in human colon cancer cells (HCT-116) inducing a significant cell inhibition, which is c...