Composite fluorescent nanoparticles for biomedical imaging

Purpose: In the rapidly expanding field of biomedical imaging, there is a need for nontoxic, photostable, and nonquenching fluorophores for fluorescent imaging. We have successfully encapsulated a new, extremely hydrophobic, pentacene-based fluorescent dye within polymeric nanoparticles (NPs) or nanocarriers (NCs) via the Flash NanoPrecipitation (FNP) process. Procedures: Nanoparticles and dye-loaded micelles were formulated by FNP and characterized by dynamic light scattering, fluorescence spectroscopy, UV-VIS absorbance spectroscopy, and confocal microscopy. Results: These fluorescent particles were loaded from less than 1 % to 78 % by weight core loading and the fluorescence maximum was found to be at 2.3 wt.%. The particles were also stably formed at 2.3 % core loading from 20 up to 250 nm in diameter with per-particle fluorescence scaling linearly with the NC core volume. The major absorption peaks are at 458, 575, and 625 nm, and the major emission peaks at 635 and 695 nm. In solution, the Et-TP5 dye displays a strong concentration-dependent ratio of the emission intensities of the first two emission peaks, whereas in the nanoparticle core the spectrum is independent of concentration over the entire concentration range. A model of the fluorescence quenching was consistent with Förster resonant energy transfer as the cause of the quenching observed for Et-TP5. The Förster radius calculated from the absorption and emission spectra of Et-TP5 is 4.1 nm, whereas the average dye spacing in the particles at the maximum fluorescence is 3.9 nm. Conclusions: We have successfully encapsulated Et-TP5, a pentacene derivative dye previously only used in light-emitting diode applications, within NCs via the FNP process. The extreme hydrophobicity of the dye keeps it encapsulated in the NC core, its extended pentacene structure gives it relatively long wavelength emission at 695 nm, and the pentacene structure, without oxygen or nitrogen atoms in its core, makes it highly resistant to photobleaching. Its bulky side groups minimize self-quenching and localization within the nanoparticle core prevents interaction of the dye with biological surfaces, or molecules in diagnostic assays. Loading of dye in the NP core allows 25 times more dye to be delivered than if it were conjugated onto the nanocarrier surface. The utility of the dye for quantifying nanoparticle binding is demonstrated. Studies to extend the wavelength range of these pentacene dyes into the near infra-red are underway.