Upconverting Nanophores
Another group of nanoparticles useful for bioimaging as well as for light activation of therapy is that of rare-earth-ion-doped oxide nanoparticles (Holm et al., 2002). The rare-earth ions are well known to produce IR to visible up-conversion by a number of mechanisms as shown in Figure 15.6. These up-conversion processes in rare-earth ions, like the two-photon absorption in organics, discussed in Chapter 5 and in Chapters 7 and 8 (two-photon bioimaging), are quadratically dependent on the excitation intensity. Thus, they provide better spatial resolution. They produce background-free (practically no autofluorescence) detection, because the excitation source is in the near-IR (generally 974-nm laser diodes). An advantage offered by these nanopar-
- Figure 15.6. Various up-conversion processes exhibited by rare-earth ions.
ticles over the two-photon dye is that the up-conversion process in the rare-earth nanoparticles is by sequential absorption through real states and is thus considerably stronger. Therefore, one can use a low-power continuous-wave diode laser at 974 nm (which is also very inexpensive and readily available) to excite the up-converted emission. By contrast, the two-photon absorption in organic dyes is a direct (simultaneous) two-photon absorption through a virtual state (see Chapter 5) that requires a high-peak-power pulse laser source. However, the emission from the rare-earth ion is a phosphorescence with a lifetime typically in milliseconds, compared to a dye fluorescence with a lifetime in nanoseconds. The concepts of phosphorescence and fluorescence have been discussed in Chapter 4. Therefore, applications that require shortlived fluorescence cannot use the phosphorescence from these up-converting nanoparticles, also referred to as nanophores or nanophosphors.
A considerable amount of work on up-converting nanophores and their applications was originally done by SRI (Chen et al., 1999). More recently, our group at the Institute for Lasers, Photonics, and Biophotonics has produced rare-earth-doped yittria (Y2O3) nanoparticles and coated them with silica to produce nanophores of size ~25nm (Holm et al., 2002). These silica-coated nanophores are water-dispersable and extremely stable and exhibit no photo-bleaching. The size of these nanophores is still small enough for them to penetrate the cell by endocytosis or by functionalizing the surface of the silica coating with a carrier group.
These nanophores are prepared using the reverse micelle chemistry described in Section 15.2 (Kapoor et al., 2000). Salts of Y and Er are used to form functionalized surfactants by replacing the cation, Na+, in the surfactant bis-2-ethylhexyl sulfosuccinate sodium salt (often abbreviated as Na-AOT). The nanophosphors are synthesized by dissolving appropriate amounts of the dried functionalized surfactant in isooctane. Particles of varying sizes can be synthesized by altering the water to surfactant ratio W0 (see Section 15.2). Then, a Na-AOT reverse micelle solution of equivalent W0 containing ammo
Figure 15.7. Bioimaging using up-converting nanoparticles on oral epithelial carcinoma cells (KB). KB cells were incubated with nanoparticles consisting of Er-doped Y2O3 nanophosphors in silica shell. Figure A represents the light transmission image of the KB cells. Figure B is the fluorescence emission after excitation with 974 nm. Figure C is the composite of Figures A and B.
Figure 15.7. Bioimaging using up-converting nanoparticles on oral epithelial carcinoma cells (KB). KB cells were incubated with nanoparticles consisting of Er-doped Y2O3 nanophosphors in silica shell. Figure A represents the light transmission image of the KB cells. Figure B is the fluorescence emission after excitation with 974 nm. Figure C is the composite of Figures A and B.
nium hydroxide is added to precipitate the hydroxide precursor nanoparticles. Encapsulation and functionalization, for subsequent ligand coupling, of nanophosphors is accomplished by addition of silica shell. The targeting ligand is then coupled to the —COOH groups or NH2 groups of the spacer arms by using carbodimides. The same procedure is followed to synthesize Er/Yb co-doped Y2O3 and Tm/Yb co-doped Y2O3. The up-converted emission of these nanoparticles is red (640 nm) for the Er/Yb co-doped Y2O3 particles, green (550nm) for Er-doped Y2O3 particles, and blue (480nm) for Tm/Yb co-doped Y2O3 particles. These wavelengths of light are readily detected with standard CCD arrays and/or a CCD-coupled spectrograph. The use of an IR laser drastically reduces the problems associated with the use of a UV excitation source.
Moreover, the ability to tailor the emission wavelength coupled with our ability to surface functionalize these nanoparticles allows for a number of unique applications of these materials. Our initial studies were conducted in the KB cells (Holm et al., 2002). As can be seen in Figure 15.7, the infrared excitation wavelength does not induce autofluorescence in the target cells. Only the fluorescence emission of the nanophores can be seen (Figure B). This signal-to-noise ratio reduction is of great benefit in the visualization of low-level fluorescent signals in biological systems.
At our Institute for Lasers, Photonics, and Biophotonics, the silica-encapsulated rare-earth-doped Y2O3 nanoparticles are also being investigated for multiphoton photodynamic therapy (Roy et al., 2003). The basic concept is similar to the one discussed in the section on two-photon photodynamic therapy in Chapter 12. However, here one utilizes the IR-to-visible up-conversion in these nanophores and not a two-photon active dye, discussed in Chapter 12. The benefits are again greater penetration into a tissue offered by the use of a near-IR (974-nm) excitation source.
For this purpose, we used a well-established photodynamic photosensitizer (PDT drug) HPPH, discussed in Chapter 12, to test the ability of the nanophos-phors to excite HPPH. The following study was performed. Sintered nanopar-
- Figure 15.8. Nanophosphor excitation of HPPH. (A) Red emitting nanophosphors, (B) green emitting nanophosphors, (C) blue nanophosphors, (D) HPPH excitation by 974 nm.
ticles were dispersed in DMSO to obtain a translucent colloidal dispersion of nanophosphors. Equal volumes of the nanophosphor solution and 1 mM of HPPH in DMSO were mixed in individual cuvettes. Identical solutions containing only HPPH or the nanoparticles were also placed into cuvettes. Each cuvette was pumped with a 974-nm CW diode laser, and the emission spectra were collected at 90° to the excitation laser with a fiber-coupled CCD spectroscope. The data were normalized to the maximum peak intensity and plotted with identical nanophosphor blank solutions.
It is clearly seen in Figure 15.8 that within the experimental parameters, both the green and red emitting nanophosphors are capable of exciting HPPH. Coupling of the emission of the nanophosphor with HPPH is shown by loss of emission by particle and appearance of the HPPH emission. The blue-emitting nanophosphors, however, did not demonstrate any significant coupling with HPPH. This lack of fluorescent emission from HPPH is due to absence of overlap of emission of the blue nanophosphors with the absorption region of the HPPH.
Figure 15.9. Schematics of a PEBBLE nanosensor, with various functions shown. Current matrix materials are presented on the right. (Reproduced with permission from http://www.umich.edu/ ~koplab/research2/analytical/EnterPEBBLEs.html.)
Acrylamide
+ Molecular probe
+ dextran + Magnetic dipoles
Acrylamide
+ Molecular probe
+ dextran + Magnetic dipoles
Liquid polymer
(PVC or decyl-methacrylate) + additives + PEG
Liquid polymer
(PVC or decyl-methacrylate) + additives + PEG
Sol-Gel
Sol-Gel
Figure 15.9. Schematics of a PEBBLE nanosensor, with various functions shown. Current matrix materials are presented on the right. (Reproduced with permission from http://www.umich.edu/ ~koplab/research2/analytical/EnterPEBBLEs.html.)
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