Journal of the American Chemical Society Here, we have developed a photoswitching nanoparticulate system that uses light as the remote means of triggering both on-demand drug release and reversible changes in particle volume to enhance tissue penetration. ■ RESULTS AND DISCUSSION Photochromic properties are controllable light-induced changes in color or reversible photoexcited transformations between two isomers.18 There has been intensive investigation of photochromic materials for applications from sunglasses to optically rewritable data storage,19 optical switching,20 and chemical sensing.21 The photoswitchable NPs developed here were composed of spiropyran (SP, a family of photochromic molecules, Figure 1a,b) and lipids. SP consists of a nitro- Figure 1. (a) Structure and photoisomerization reaction between spiropyran (SP) and merocyanine (MC). (b) Abbreviations for SP and MC derivatives. (c) Scheme of photoswitching SP NPHs composed of SP-C9 and DSPE-PEG. Yellow oval, SP molecule; blue line, the alkyl chain (R) in SP; red, lipid part; green line, PEG. SP NPHs are converted to MC NPHs (purple sphere: MC molecule) by UV light irradiation; the reversible photoisomerization from MC NPHs to SP NPHs happens in dark but is accelerated by visible light (500−600 nm). benzopyran and an indoline moiety with orthogonal orientation (Figure 1a). Both moieties absorb in the ultraviolet spectrum independently.22 Ultraviolet light (UV, 365 nm) induces ring- opening in the pyran to form merocyanine (MC, Figure 1a). The nitrophenol and indoline chromophores are merged to form one large planar π-system, leading to intense absorption in the visible (Vis) spectral region (500−600 nm).23 The zwitterionic MC form is less stable than the hydrophobic SP form and undergoes spontaneous ring-closing back to SP in the dark that is accelerated by photoexcitation of MC in the Vis absorption band.18a The polarity or hydrophilicity changes of SP molecules that accompany their photoisomerization have been suggested to alter microenvironments within polymers and supermolecular assemblies such as Langmuir−Blodgett films, micelles, and liposomes.20b,24 We hypothesize that SP isomerization upon irradiation would lead to hydrophilicity changes which would switch the NPs’ physical assembly properties and trigger drug release. Of note, micromolar concentrations of SP derivatives are reported to have minimal cytotoxicity in macrophages, gastric cells, and epithelial cells after exposure for 72 h.25 These properties suggest that SP is a suitable base material for light-responsive NPs for triggered release. Formulation of Photoswitchable NPs with Light- Triggered Size Changes. SP derivatives bearing hydrophobic alkyl chains (Figure 1a,b) were synthesized by coupling 2- Article hydroxy-5-nitrobenzaldehyde with substituted 2,3,3-trimethyl- 3H-indolium iodide (Figure S1a). NPs were initially prepared by direct nanoprecipitation of SP alone (an extensively used simple method for the preparation of NPs with therapeutic agents embedded in the hydrophobic matrices).26 An acetonitrile solution of SP-C9 (10 mg/mL) was nano- precipitated into water (final acetonitrile/water = 1/40, v/v), resulting in NP sizes of 198.1 ± 2.5 nm with a polydispersity of 0.09 ± 0.02, determined by dynamic light scattering (DLS, N = 5, Table S1). Irradiation of the SP-C9 NPs with UV light (365 nm, intensity ∼1 W/cm2, ∼ 3.1 × 10−6 einstein) led to photoisomerization and the subsequent conversion of hydro- phobic SP-C9 to amphiphilic MC-C9, and a change in the sizes of the NPs. The irradiated NPs had a bimodal size distribution (Figure S1b), with one peak at 39.6 ± 3.0 nm (N = 5, 99.1% of number population, determined by DLS; attributable to NPs assembled by MC-C9), and another at 202.1 nm (0.9% of number population; attributable to NPs formed with SP-C9). After irradiation, the colorless NP solution became purple, with a strong Vis absorption band characteristic of MC-C9 (maximum absorption wavelength λmax = 560 nm; Figure S1c,d). Nanoprecipitation of a SP analogue with a shorter alkyl chain, SP-C7, produced NPs that did not undergo a significant size change upon UV irradiation (Table S1). SP-C9 NPs formed in aqueous solution aggregated when introduced into PBS (Table S1), presumably due to salt- induced screening of electrostatic repulsive forces between particles.27 In addition, the NPs had low actual drug loadings wt % (loading wt % < 1%) and efficiencies (<13%; Table S2). The loading efficiency did not increase in NPs made of SPs with a longer alkyl chain (SP-C18, Table S2). Higher drug loading of delivery vehicles is desirable for optimal therapeutic effect, to enhance the potency of NPs that reach the tumors.28 To improve the stability and loading efficiencies of NPs while maintaining the NPs’ photoswitching properties, we produced hybrid SP/lipid-polyethylene glycol (PEG) NPs (termed NPHs; Figure 1c) using a rapid ultrasonication method.29 An acetonitrile solution of SP-C9 (1 mg/mL) was slowly added into a 4 wt % ethanolic aqueous solution containing lecithin and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- carboxy(polyethylene glycol)-5000 (DSPE-PEG, [SP-C9]/ [DSPE-PEG]/[lecithin] = 32/16/1), followed by addition of water to adjust the organic/aqueous solution volume ratio to 1/ 10. After sonication for 8 min and filtration of the organic solvent, SP NPHs were obtained with an average hydrodynamic diameter of 143.2 ± 2.1 nm and a polydispersity of 0.03 ± 0.01 (N = 5, Figure 2a). SP-C9 was not detected by HPLC in the filtrate after repetitive washing of the NPHs by ultra- centrifugation, indicating that SP-C9 was completely incorpo- rated into NPs (Figure S2a,b). After UV illumination (30s, ∼100% conversion to MC), the absorption band of the NPHs moved to a λmax at 551 nm (Figure 2b). As with the nonhybrid NPs, UV irradiation of NPHs induced a size change (to 47.1 ± 1.3 nm, polydispersity of 0.05 ± 0.02, N = 5). These results confirmed that both the photochromic properties of SP-C9 and light-triggered size change were maintained in the SP NPHs. MC NPH reverted to SP NPH in darkness or by Vis light, with an accompanying increase in volume (Figure 2a). Consequently, there could be inaccuracies in measuring MC NPH size by relatively slow techniques such as DLS. To confirm particle shrinkage after irradiation (Figure 2a), we produced NPH containing MC−CN, a similar but relatively stable MC 8849 dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855