Journal of the American Chemical Society Article Figure 5. (a) Flow cytometric analysis of the internalization of Cy5 in SP NPM-Cpp. Red line, untreated HeLa cells; green line, HeLa cells treated with Cy5/SP NPM for 30 min; blue line, HeLa cells treated with Cy5/SP NPM-Cpp for 30 min; (b) MTT assay to determine the differential cytotoxicity of doxorubicin/SP NPM, and doxorubicin/SP NPM. Data are means ± SD, N = 6, asterisks indicate P < 0.005. than the doxorubicin/SP NPM (Figure 5b). These results suggest that the SP NPM’s have the capacity to be functionalized by a broad range of biomolecules (e.g., aptamers40 or other peptides41) to enhance drug delivery. Light-Triggering Enhances Diffusion in Collagen Matrices. As discussed above, the ability to penetrate tissue could have a bearing on therapeutic effectiveness. We evaluated whether the light- triggered size change could enhance diffusive transport through a dense collagen gel at a concentration (0.74%; 7.4 mg/mL12) similar to the 9.0 ± 2.5 mg/mL of interstitial matrix estimated for interstitial collagen in human tumors (e.g., LS174T) and murine tumors (e.g., MCalV).10b,42 SP NPHs (1 mg/mL) loaded with 5 wt % indocyanine green (ICG), a NIR dye, were placed in contact with collagen gels in a horizontal capillary tube, then incubated for a further 12 h at 37 °C. A NIR imaging system was used to track particle infiltration into the collagen (Figure 6). Free ICG penetrated Figure 6. Normalized NIR fluorescence intensity vs distance profiles for various formulations’ penetration into collagen gels over a period of 12 h (7.4 mg/mL): free ICG (black, diffusion coefficient = 3.59 ± 1.94 × 10−7 cm2·s−1), ICG/SP NPH (red, diffusion coefficient = 7.65 ± 1.63 × 10−7 cm2·s−1), ICG/SP NPH irradiated by UV light (blue, diffusion coefficient = 2.24 ± 0.42 × 10−6 cm2·s−1 at t = 0 for 10 s), and ICG/SP NPH irradiated twice by UV (green, for 10 s each time, separated by 3 h,). Diffusion coefficient data are means ± SD, N = 4. The dashed lines are theoretical curves fitting the intensity profiles using a one- dimension diffusion model. 4.0 ± 0.21 mm into the collagen gels, ICG/SP NPH penetrated 8.3 ± 0.10 mm without UV triggering, and ICG/SP NPH triggered by UV for 10 s penetrated 12.1 ± 0.02 mm (N = 4, P < 0.005 for irradiated ICG/SP NPH compared to free ICG and unirradiated ICG/SP NPH). (The mechanical properties of collagen barely change after 1 h irradiation at 254 nm UV light, ∼1.7 × 10−6 einstein.43) By fitting the fluorescence intensity of ICG/SP NPH to a one-dimensional diffusion model, we obtained an average diffusion coefficient of 2.24 ± 0.42 × 10−6 cm2·s−1 for UV-triggered ICG/SP NPH NPs (N = 4, P < 0.005 compared to free ICG), while the diffusion coefficient for unirradiated ICG/SP NPH (7.65 ± 1.63 × 10−7 cm2·s−1, N = 4) was not statistically significantly different from that of free ICG (3.59 ± 1.94 × 10−7 cm2·s−1, N = 4, P = 0.064) compared to unirradiated ICG/SP NPH (Figure 6). The relatively low diffusion rate of free ICG in collagen gels compared to NPHs might be partly due to the lipophilicity of ICG.44 Gel penetration was further enhanced by increasing irradiation: ICG/SP NPH irradiated twice (for 10 s each, separated by 3 h) penetrated 16.8 ± 0.10 mm with an average diffusion coefficient of 1.97 ± 0.28 × 10−6 cm2·s−1 (calculated by modified one dimension diffusion models; N = 4; Figure 6 green line). The fact that the diffusion coefficient of light- triggered ICG/SP NPH was significantly larger than those for nonirradiated ICG/SP NPH and free ICG (for both P < 0.005) suggests that light-induced shrinkage might help deepen tissue penetration of SP NPH and their payloads. That possibility is supported by the observation that irradiation does not appear to affect the other physicochemical properties of PEGylated NPH (they have similar slightly negatively charged surfaces before and after irradiation). Enhanced Diffusion of Photoswitching NPs in the Cornea. We assessed the potential for photoswitching SP NPH to carry drugs across the cornea in a manner analogous to the findings in collagen gels. Corneas are composed of 90−95 wt % of dense collagens, rendering the delivery of drugs through the 8852 dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855