Journal of the American Chemical Society cornea to the anterior chamber difficult. Particles containing Cy5 (Cy5/SP NPH) were applied to fresh cadaveric porcine corneas with or without UV light triggering for 1 min, and incubated for 8 h. Gross examination of the corneas and NIR scanning of Cy5 in corneal cross section demonstrated that the diffusion of Cy5/SP NPH was markedly enhanced by UV light triggering (Figure 7). Histologically, corneas treated with Cy5/ Figure 7. Ex vivo study of Cy5/SP NPH penetration in porcine corneas. (a) Fresh corneas after an 8-h treatment with Cy5/SP NPH (with or without UV irradiation for 1 min) or Cy5. The green color indicates the presence of Cy5 (a blue dye that becomes greenish in the slightly yellow tissue of the eye); (b) near-infrared images of cross sections of corneas tissues treated as in panel (a). The scale bar = 1 cm. SP NPH and UV light were indistinguishable from untreated controls under light microscopy, showing no tissue injury (Figure S9). Since collagen is one of the major components of the interstitial matrix, these results suggest the potential usefulness of SP NPH for light-triggered drug delivery to targeted tissues, for example, eyes and tumors. These results are consistent with a recent report that polymeric micelles ∼30 nm (close to MC NPHs sizes) showed enhanced tissue penetration and potent antitumor activity in poorly permeable pancreatic tumors.45 The histological findings, together with the benign cytotoxicity (Figure S5) are consistent with a favorable safety profile, but this remains to be validated by in vivo studies. The wavelengths of the UV light we used for SP NPH triggering might limit the application of this technology to areas of the body that can be illuminated directly, for example, the eyes and ears. Of note, the photochromic conversion of SP could be potentially triggered at depths up to several centimeters by near-infrared lasers using two-photon technol- ogy (wavelength ∼ 720 nm), through soft tissues, bone, and intact skull.46 Fluorescence of Photoswitching NPs. The possibility that NPH could perform as fluorescent light-triggered imaging probes was suggested by the fact that SP or nanoparticles surface-modified with SP have been utilized as fluorescence imaging probes in different microscopy techniques, including optical lock-in detection (OLID),27 two-photon photoswitch- ing, and imaging by noninvasive near-infrared (NIR) light.28 Article Although MC-C9 does not fluoresce in organic solvents (e.g., acetonitrile), we found that NPH could switch between fluorescence (as MC-C9) and nonfluorescence (as SP-C9). UV-irradiation of SP NPH in aqueous solution created MC NPH (Figure 1c) with an ∼8-fold increase in red fluorescence (600−800 nm) compared to MC-C9 in acetonitrile ([MC-C9] = 0.20 mM for both acetonitrile solution and NPHs). The λmax of MC NPH red-shifted by 32 to 672 nm compared to MC-C9 in acetonitrile (Figure S10a and associated discussion of mechanism). The fluorescence exponential decay constant of MC NPH (Figure S10b) was 1.44 × 10−4 s−1 at 672 nm (t1/2 = 4813 s), much slower than for free MC-C9 in acetonitrile solution (t1/2 = 346 s). The intensity of the fluorescence and the duration of the decay of that intensity for MC in NPH would be sufficient for use in microscopic imaging, unlike free MC. The fluorescent photochromic properties of NPs could be used to track them in biological studies (e.g., intracellular drug delivery) with greater reliability than with simple fluorescence, which can be confounded by interfering fluorophores or in vivo autofluorescence.46c,47 In fact, NPs surface-modified with SP have been utilized as light-triggerable fluorescent probes.24h Here, we evaluated whether fluorescence switching of SP NPH could be achieved in living cells in vitro in a HeLa cell line (Figure 8a). We loaded Cy5 (emission max = 690 nm) into SP Figure 8. Fluorescence images of the internalization of Cy5-containing NPH by HeLa cells after 2 h incubation. (a) Nuclear staining with DAPI (blue color). (b) NPHs were illuminated by UV for 2s then imaged with 560 nm emission filters (green color); NPHs were seen to be internalized. (c) The red color (emission at 700 nm) shows the Cy5 loaded in the SP NPH. (d) The overlay of panels a−c. The orange color demonstrated colocalization of SP NPH with Cy5. The scale bar = 50 μm. NPH since its emission spectrum would have little overlap with that of MC NPH (emission max = 672 nm). Cy5-containing SP NPHs were incubated with HeLa cells for 2 h in darkness then exposed to UV illumination for 2 s, causing immediate fluorescence attributable to MC NPH (Figure 8b). Fluorescence microscopy (Figure 8) indicated that Cy5 (red color) and MC- containing hybrid NPs (green color) were colocalized in HeLa cells (orange color in Figure 8d). Mechanism of Photoswitching NPs. We propose the following assembly model to explain the photoswitching of SP NPH (Figure 9). The SP NPHs are composed of a hydrophilic 8853 dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855