Journal of the American Chemical Society m2).36 The EC50 value for MC NPH in HeLa cells was 3.46 mg/ mL, similar to that for SP NPH (Figure S5b). Repetitive Photoswitching and Light-Triggered Drug Release Profiles of NPs. The repeatability of the photo- switching property of NPH was evaluated by alternating cycles of UV and Vis light. This modulation was fully reversible for at least 4 continuous cycles (UV irradiation for 30 s and Vis light for 3 min, Figure 3). However, the absorbance at the MC-C9 Figure 3. Reversible NPH photochromism (solid line, Abs: absorbance) and size switching (dashed line) with alternating UV (“UV”, 30 s) and visible light (“Vis”, 3 min) irradiation. The modulation of NPH size and photochromism was fully reversible for at least 4 cycles. Data are means ± SD, N = 4. peak maximum decreased 43% after 4 cycles, and was accompanied by a reduction in size from 143.2 to 98.7 nm in the SP state (Figure 3). The decrease of absorbance after repetitive irradiation could be due to photofatigue (the loss of performance in photoisomerization)  a common property of organic photochromic compounds.37 The absorption intensity of MC in NPH at 551 nm faded at a rate dependent on the UV (365 nm) irradiation time, and that antioxidant agents could not eliminate the decrease in MC-C9 absorption, suggesting an O2-independent fatigue mechanism for photofatigue in SP NPHs (see Figure S6 and Scheme S1 and associated discussion). We hypothesized that the phototriggered shrinkage of NPHs might induce drug release. In the absence of UV photo- triggering, drugs (e.g., doxorubicin) and dyes (e.g., rhodamine 6B) loaded in SP NPH showed slow release in PBS that was complete within 48−72 h (Figure 4, Figure S7). Upon UV irradiation (30s), NPHs encapsulating rhodamine 6B (loading wt% = 4.3%) released 29.3% of the loaded dye within 1 h as determined by HPLC, while 7.2% was released in the same period without UV irradiation. Of note, the release kinetics of NPHs that had been triggered (Figure 4, blue line) eventually slowed to a rate similar to that of NPHs that were not irradiated (Figure 4, black line). This decrease in the release rate could be explained by the majority of the MC-C9 in NPs spontaneously converting back to SP-C9, resulting in NPs reassembled in their original structure. In a separate group, UV triggering (30 s irradiation) was conducted every 3 h for three cycles (Figure 4, green line), with an increase in release at each event. UV-triggered release was demonstrated in cells by fluorescence imaging of SP NPH loaded with calcein. Calcein was selected because its fluorescence self-quenches while it is entrapped inside particles, whereas calcein released from Article Figure 4. Release profiles in PBS for rhodamine B loaded in SP NPH under different conditions: without irradation; with UV irradiation for 30 s at 0 h; with repetitive UV irradiation at 0, 3,and 6 h. The times of irradiation are indicated by purple arrows. Data are means ± SD, N = 6. particles will become diluted and fluoresces.38 SP NPH loaded with calcein (2.7 wt %) were incubated with HeLa cells. After 4 h, the media containing NPHs was removed and the cells were washed with PBS. Cells in medium were then illuminated by UV (365 nm) for 2 s, left in darkness for 5 min, then imaged (Figure S8). Strong fluorescence intensity with an emission maximum at 510 nm was noted in the cells, indicating that the calcein was released from NPs that had been taken up. Illumination followed by imaging was repeated 5 times, during which the fluorescence intensity gradually increased to saturation (Figure S8a,b). Cells treated with same NPs but without UV irradiation did not fluoresce, suggesting that the UV triggered rapid calcein release and intracellular dispersal from SP NPH. These results were validated by flow cytometry, which showed a 24.7-fold increase in fluorescence intensity after a 10 s UV treatment (Figure S8c). Surface Functionalization of NPs. Nanoparticle ther- apeutic effect can be enhanced and toxicity reduced by surface modification with moieties that allow intracellular penetration and/or targeting of specific tissues.39 To examine the potential suitability of the NPH for targeted drug delivery, we formulated NPs (NPM) composed of SP-C9 and a mixture of DSPE- PEG3400-maleimido (DSPE-PEG-MAL) and DSPE-PEG in a 4/ 1 ratio (w/w), 153.1 nm in diameter and with a polydispersity of 0.09. A cell penetration peptide (Cpp) Cys-Tat (47−57) (sequence: CYGRKKRRQRRR-NH2) was introduced onto SP NPM loaded with Cy5 by reaction of the carboxyl-terminal Cys of the peptide with the MAL on the NPM surface (NPs/Cpp = 100/1, w/w). The fluorescence intensity of HeLa cells incubated with the resulting NPs (SP NPM-Cpp) for 30 min, measured by flow cytometry, was 7.1 times higher than that of cells treated with SP NPM lacking Cpp (N = 4, fluorescence intensities of 1940 ± 215 and 273 ± 197, respectively; Figure 5a). We compared the cytotoxicity of doxorubicin-loaded SP NPM-Cpp (doxorubicin/SP NPM-Cpp) to that of SP NPM without Cpp (doxorubicin/SP NPM). HeLa cells were incubated with doxorubicin/SP NPM-Cpp or doxorubicin/SP NPM for 2 h, then incubated in medium without NPs for a total of 48 h; cell viability was measured by MTT assay. The doxorubicin/SP NPM-Cpp were significantly more cytotoxic 8851 dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855