Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells

Abstract

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50–60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol–gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230–260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.

Functionalization of Photosensitized Silica Nanoparticles for Advanced Photodynamic Therapy of Cancer

“…For the samples RB-PEG2000-NP-a, RB-PEG5000-NP-a, and RB-PEG-NP-c (RB grafted at the hydroxyl groups of MSNs, Table 2) the estimated RB loading was equal and consequently, the observed dye aggregation in these samples should be assigned to interparticle processes, as supported by Zpot values and previously attributed to a lower presence of PEG molecules at the external surface. Sample RB-PEG-NP-b, with RB loading at the amine groups half of that obtained for the RB at the hydroxyl groups (sample RB-PEG-NP-b vs sample RB-PEG-NP-a in Table 2), showed a narrower absorption band, not much different from that recorded for RB in diluted solution, except for the typical blue-shifted and slight broadening found in dyes in adsorbed-state [80]. However, reducing the cargo of PS per nanoparticle would compel a higher concentration of nanoparticles per volume to reach effective PS doses for PDT in the cells, which would also promote particle-particle agglomeration.…”

“…Mesoporous silica nanoparticles with a suitable size for medical applications and particularly for PDT [73,89], were synthesized by the modified Stöber method [90] as described elsewhere [80]. The external surface of mesoporous nanoparticles surface was functionalized with amino group (NH-MSN) or carboxylic group (COOH-MSN).…”

“…Thus, tethering FA on nanoparticle's surface promotes a higher cellular uptake via endocytosis [76][77][78][79]. In fact, our recently published study has quantitatively demonstrated a significantly higher accumulation of FA-functionalized fluorescent MSNs compared to nanoparticles without FA in HeLa cells [80].…”

“…The synthesis of mesoporous silica nanoparticles with amine groups at the external surface (NH-MSNs) has been described previously [80,81]. The synthesis route of mesoporous silica nanoparticles with carboxylic group (COOH-MSN) is similar to that described for NH-MSN but in the second step CTES (0.007 mmol) is added instead of APTMS to provide cyane groups at the external surface.…”

“…-RB-PEG-NP-b: RB was linked through its carboxylic group to the amine groups of NH-MSN (40 mg) by carbodiimide method. This method was previously described [80,81]. Briefly, NHS/EDC is added to RB in solution (0.03 mmol in 20 mL of CH3CN) in equimolar concentration and stirring for 1 h in an inert atmosphere.…”