(6f) Folate Mediated Delivery of Dye-Doped Silica Nanoparticles for Biolabelling

The efficient clinical management and understanding of cancer biology requires a highly sensitive detection system to image tumors at the very onset. This should presumably lead to faster and more effective treatment of patients. Imaging techniques have continued to develop in recent years, to facilitate the screening for cancer. Ideally, cancer-imaging agents should have excellent specificity towards biomolecules of interest and have an optically stable signal transduction. The use of bioconjugated fluorescent silica nanoparticles (FSNPs) offers a non-toxic, photostable system that can be used for attaching target specific molecules. In the past, folates have been extensively utilized as delivery vehicles for transporting imaging or therapeutic agents to certain cancer cells. Covalent coupling of folate to various molecules yields a conjugate that can be endocytosed by cancer cells. Following this strategy, radiopharmaceutical agents, chemotherapeutic agents and antisense oligonucleotides have been successfully delivered inside tumors. It has been reported that lung adenocarcinoma overexpress folate receptors. Using folate receptor mediated delivery mechanism, it is likely that folate conjugated fluorescent nanoparticles is delivered into lung cancer cells. In an attempt to demonstrate the feasibility of the concept, we have used 150 nm folate conjugated fluorescent silica nanoparticles for targeting A549 lung cancer cells.

Methodology

150 nm monodispersed FITC doped nanoparticles was synthesized using Stobers' method. The silane precursor, FITC-APTS, was first prepared in absolute ethanol using an excess amount of APTS, to obtain additional free amine groups on the dye surface. Briefly, 800 ml NH4OH was combined with 10 ml absolute ethanol and stirred on a magnetic plate followed by the addition of 400 ml TEOS and 200 ml of the FITC-APTS conjugate after a few minutes. 60 ml THPMP was added to the solution after 30 min and magnetic stirring was continued for 24 hours. The negatively charged silane agent, THPMP improved the aqueous dispersibility of amine-modified FSNPs. The excess APTS used to synthesize the FITC-APTS conjugate was two fold, first, to ensure that all FITC molecules had reacted, and second, to directly obtain a coating of primary amine group on the nanoparticle surface for ease of bio-conjugation. For folate conjugation of the FSNPs, the nanoparticles were centrifuged and thoroughly washed with ethanol (three to four times) and DI water (two to three times). An ultrasonic bath was used to re-disperse the nanoparticles in solution, after each centrifugation. Folate immobilization was done using the water-soluble carbodiimide (WSC) coupling chemistry. Two solutions were prepared and stirred for 20 min. Solution I (4.0 ml, 25% DMSO in water) contained 7.0 mg folate in 1.0 ml DMSO, 4.1 mg NHS in 1.5 ml DI water and 34.2 mg EDC in 1.5 ml DI water. Solution II contained amine-FSNPs (20 mg in 1.0 ml DMSO) and triethylamine (2.0 ml). The solutions were combined and sonicated for 5 min, and stirred magnetically for 1 h. Folate conjugated FSNPs were centrifuged and washed five times with DI water and protected from light until further use.

Results

FSNPs were characterized using a variety of microscopic (TEM), light scattering (Coulter LS13 320) and spectroscopic (UV-Vis and fluorescence) techniques. Based upon the size (diameter) measurement of 100 particles, the average nanoparticle size obtained was 150 nm (+5 nm standard deviation). TEM images showed that the FSNPs were monodispersed. The presence of folic acid was confirmed by spectroscopic methods. Steady-state fluorescence excitation and emission spectra were recorded on a spectrofluorometer to confirm the presence of both folic acid and FITC in amine-FSNPs in DI water measured at room temperature. To demonstrate the suitability of using FSNPs for cancer detection, 150 nm folate conjugated FSNPs were used to target A549 cancer cells (overexpress folate receptors) and a laser scanning confocal microscope (LSCM) was used for imaging. Human dermal fibroblast cells (do not overexpress folate receptors) were used as control cells. Most of the particles were found to adhere on the surface of the cells while some were internalized (endocytosed) as observed from intra-cellular fluorescence. As expected, nearly no labeling was observed in human dermal fibroblast cells.

Conclusions

Folate conjugated, 150 nm fluorescent silica nanoparticles have been successfully delivered into A549 human lung cancer cells. LSCM images clearly showed endocytosis of the fluorescent nanoparticles. This research work clearly demonstrates the feasibility of developing fluorescent nanoparticle based early lung cancer detection system. We hypothesize that folate conjugated fluorescent nanoparticles could similarly be delivered to the lung by an inhaler. A bronchoscope with suitable optical filters can detect fluorescence from the labeled cancer cells. Unbound particles will eventually be phagocytosed and cleared by alveolar macrophages. Although silica nanoparticles have been used in the present work as a model system, silica can be replaced with a suitable biodegradable material and can be loaded with both imaging. In the future, we would like to use this detection system for the detection of other types of cancer cells/cancer tissues that overexpress folate receptors.