Journal of the American Chemical Society Article in PBS (Figure S2c). The stability of NPHs was also evaluated in serum by monitoring the absorbance change at 560 nm, since nanoparticles cannot be accurately detected in dense serum solutions by DLS.32 No significant aggregation was observed over 4 h. For eventual clinical translation, it has to be possible for NPs to be stable during manufacturing, storage, and trans- portation.33 SP NPHs were lyophilized for 48 h with bovine serum albumin (BSA, NP/BSA = 1/15, w/w), a known lyoprotectant reagent,34 then stored at −20 °C for over one month. The subsequent reconstitution of lyophilized SP NPH in PBS did not significantly change the NPH sizes and photochromic properties (Figure S4). Lyophilization of SP NPH in water (without albumin) led to micrometer-sized, nondispersible aggregates upon reconstitution in PBS. Since albumin is used clinically, this lyoprotection strategy may be useful for potential translational of SP NPs. To examine whether this formulation could be used to form NPs containing a broad range of compounds, we tested the ability to encapsulate rhodamine B, coumarin 6, cyanine 5 (Cy5), paclitaxel, docetaxel, proparacaine, and doxorubicin. NPs with adjustable loadings up to 10 wt % (with relatively high loading efficiencies) and low polydispersities were readily obtained for all of the therapeutics (docetaxel, doxorubicin, proparacaine) and dyes (Cy5, rhodamine B, coumarin 6) tested (Table 1). HeLa (cervical cancer cell), PC-3 (human prostate carcinoma), and human umbilical vein endothelial cells (HUVEC) were used to assess the cytotoxicity of SP NPHs. Following 72 h of exposure to NPs, cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.35 The SP NPH did not cause significant cytotoxicity in either cell line except at extremely high concentrations (Figure S5a). The EC50 values (the concentrations at which cell viability was reduced by 50%, determined by interpolation from the data in Figure S5a) for the [SP-C9] in those NPHs were 9.53 mM for HUVEC (6.33 mg/mL NPHs), 7.01 mM for HeLa cells (4.66 mg/mL NPHs), and 7.41 mM for PC-3 cells (4.92 mg/mL NPHs). In a 70 kg adult, these EC50 values are approximately equivalent to 70 g/ dose (∼ 1 g/kg) assuming NPHs are restricted to the 14 L extracellular fluid, or 25 g/dose (∼350 mg/kg) if the NPs are restricted to the 5 L bloodstream, extremely high doses compared to those used clinically with Doxil (dosage: 50 mg/ 0.054 0.013 0.064 0.071 0.052 0.088 0.039 0.035 0.074 0.049 0.071 74.2 ± 2.6 27.2 ± 4.5 50.6 ± 4.8 72.7 ± 0.8 40.1 ± 8.9 76.1 ± 5.2 49.7 ± 5.8 41.5 ± 6.4 49.8 ± 6.7 48.2 ± 5.4 66.1 ± 2.5 0.081 0.086 0.072 0.066 0.065 0.066 0.043 0.043 0.058 0.100 0.032 49.8 68.4 54.2 94.1 79.4 82.1 74.2 53.7 49.6 63.5 51.0 size (nm) 129.7 ± 1.8 74.7 ± 2.9 133.9 ± 6.7 108.6 ± 4.5 101.7 ± 3.1 116.1 ± 1.1 125.4 ± 5.0 96.9 ± 4.7 93.3 ± 3.2 87.5 ± 2.7 102.3 ± 6.6 Figure 2. (a) Dynamic light scattering measurement of size changes of SP-C9/DSPE-PEG/lecithin SP NPHs upon alternating UV (30 s) and visible light (3 min) illumination. Inset: the solution of NPH before and after UV irradiation. (b) Steady-state absorption spectra of NPH ([SP-C9] = 0.46 mM) and their corresponding isomerized MC NPH (λmax = 551 nm) upon UV light irradiation. quinoidal structure, which is a 1, 6-addition adduct of MC-C9 with trimethylsilyl cyanide (Figure S3).30 MC−CN NPHs were 59.4 nm in diameter, with a polydispersity of 0.04 (Figure S3c), similar to the size of MC NPH produced from SP NPH by UV- irradiation (47.2 nm with a polydispersity of 0.05, Figure 2a). Direct nanoprecipitation of MC−CN resulted in 42.6 nm NPHs with a polydispersity of 0.11 (Figure S3d), a result consistent with the DLS measurements of MC NPHs. The PEGylated lipid was designed to give NPHs a relatively neutral surface charge for prolonged circulation and stabiliza- tion.31 The ζ potential of SP NPH and MC NPH at pH 7.5 was −6.25 ± 0.31 mV and −5.12 ± 0.12 mV, respectively. The results indicated the similarly neutral charges of NPH before and after irradiation. No aggregation was observed for over 4 h Table 1. Characteristics of Photoswitching SP NPHa drug/dye Rhodamine B Coumarin 6 Calcein Cyanine 5 Paclitaxel Paclitaxel Docetaxel Doxorubicin Doxorubicin Proparacaine Proparacaine 5 10 5 10 5 10 10 5 10 10 15 actual LD % 2.49 ± 0.13 6.84 ± 0.07 2.71 ± 0.09 9.41 ± 0.05 3.97 ± 0.04 8.21 ± 0.14 7.42 ± 0.11 2.69 ± 0.21 4.96 ± 0.14 6.35 ± 0.16 7.64 ± 0.19 initial LD % LD efficiency % polydispersity size-UV (nm) polydispersity aDetermined by DLS and HPLC. Abbreviations: LD, loading; size-UV, sizes of NPs treated by UV irradiation (N = 5). Data are means ± SD (N = 5). 8850 dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855