Nanoparticles Engineering towards Targeted Anticancer Drug Delivery and Bio-Sensing

Despite all research efforts over the past decades, cancer remains one of the main causes of death worldwide. With the results reported in this thesis, I give my contribution to fundamental research on cancer by both proposing a novel drug delivery system (DDS), which presented several advantages compared to the existing nanocarriers and by introducing innovative spectroscopic methods for cancer-related investigations.

Polymeric surface functionalization of nanoparticles has attracted much attention in the development of drug delivery systems (DDSs), as being an efficient and relatively easy engineering method to promote specific internalization of particles inside target tumor cells.  However, techniques towards the control of the intracellular route, localization and retention after the nanoparticle uptake are still limited, inhibiting the accomplishment of high performance at low drug dose. In this part of the thesis, a simple method to develop highly efficient DDSs based on polymeric bilayer functionalization of mesoporous silica nanoparticles was proposed. This delivery system combines, for the first time, the tumor targeting and the control of the intracellular route, promoting an endosomal escape effect. The active targeting was confirmed by estimating the uptake rate of the DDS in cancer and normal cells, respectively. The endosomal escape was verified by following the cellular distribution of dye-loaded particles overtime. Furthermore, controlled drug release and viability tests demonstrated that this hybrid strategy is highly efficient towards cancer cells. The high killing effect, combined with the targeting capability, the control of the intracellular route and the well-controlled drug release, makes this hybrid DDS be extremely promising for enhancing the chemotherapy efficiency and reducing the side-effects in vivo .

During in cellulo drug release experiments via fluorescence microscopy, the emission quenching of the compound upon the intercalation between the DNA base-pairs in the nucleus made the detection of the drug cellular distribution and the understanding of the action mechanism extremely problematic. In this section of the thesis, I further investigated this fluorescence quenching by incubating the drug with living cells, confirming that the same phenomenon occurs by using drug molecules dispersed in solution (not encapsulated in the DDS). Consequently, I proposed an alternative technique to fluorescence towards drug distribution studies via Raman spectroscopy. Our group previously had reported an innovative tool for intracellular investigations with high spatial resolution based on silver nanowire-mediated SERS (surface enhanced Raman scattering) endoscopy. Here, I advanced the previously-reported probes for enhancing the SERS activity of the nanowire towards the detection of drug traces inside the nucleus. The enhancement of the SERS activity was obtained by inducing the formation of gold nanostructures on the crystalline surface of silver nanowires. The high efficiency of the probe was demonstrated by collecting SERS spectra from a conventional Raman reporter. The application of the advanced SERS endoscopy for detecting drug distribution was verified by collecting SERS spectra from the nucleus of a single cell treated with an anticancer drug. This study demonstrated the potential of this technique as alternative method to fluorescence microscopy in intracellular bio-sensing applications.

Fundamental studies of cancer cell plasma membrane have been hot-topic among cancer researchers worldwide over the last few decades, as being the key basics for the development of new cancer targeting technologies. SERS spectroscopy might be an ideal tool for such investigations; however its use is limited by the challenging and time consuming fabrication of SERS substrates. In order to overcome this issue, I developed new simple approaches for preparing SERS substrates, suitable for cell membrane investigations. I believe that these approaches, thanks to its facility and short-time procedure, could be adopted by any kind of researchers and can promote breakthroughs on plasma membrane studies. The first fabrication introduced consists on the synthesis of gold nanoparticles by mixing reaction solutions on a polymeric surface at room temperature for few minutes and rinsing. The efficiency of the substrates is maximized by the adhesiveness and flexibility of the polymeric layer. Unlike conventional solid platform, the polymeric film can adhere to a target surface and increase the SERS performances. The high plasmonic activity was verified by detecting SERS signal of pesticide traces from apple skin, demonstrating that this substrate could be a good candidate for SERS-sensing on target plasmonically inactive surfaces, such as the plasma membrane.

Because of their different optical properties, gold or silver can be selected as nanoparticle composition material depending on the applications, however silver is often preferred due to the highest SERS activity, especially at visible light frequency. In this regard, I propose a similar quick synthesis of silver nanoparticles on the same polymer and the optimization of the substrates by a gold film deposition. The gold film is meant to provide oxidation resistance and increase the biocompatibility. This hybrid structure was tested as SERS substrate. The SERS sensitivity of silver was proven by performing pH-sensing measurements and the gold layer protection was demonstrated to avoid oxidation by successfully performing SERS tests on aged substrates.  This hybrid probes could be ideal for cellular investigations thanks to the combination of the high efficiency of silver and the reasonable biocompatibility of gold.