Photoswitchable Nanoparticles for Triggered Tissue Penetration and Drug Delivery Rong Tong,���,��� Houman D. Hemmati,��� Robert Langer,��� and Daniel S. Kohane*,��� ���Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ���Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Children���s Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States * S Supporting Information ABSTRACT: We report a novel nanoparticulate drug delivery system that undergoes reversible volume change from 150 to 40 nm upon phototriggering with UV light. The volume change of these monodisperse nanoparticles comprising spiropyran, which undergoes reversible photoisomerization, and PEGylated lipid enables repetitive dosing from a single administration and enhances tissue penetration. The photo- switching allows particles to fluoresce and release drugs inside cells when illuminated with UV light. The mechanism of the light-induced size switching and triggered-release is studied. These particles provide spatiotemporal control of drug release and enhanced tissue penetration, useful properties in many disease states including cancer. ��� INTRODUCTION Controlled release technology is expected to have a profound impact in many medical fields including oncology.1 The incorporation of chemotherapeutic agents in nanoparticle (NP) delivery vehicles has improved drug solubility, reduced clearance, reduced drug resistance, and enhanced therapeutic effectiveness.2 With controlled release NP systems, a single dose can sustain drug levels within the desired therapeutic range for long periods in various diseases (e.g., diabetes3 or cancer4). Several nanoparticulate therapeutics, for example, Doxil (���100 nm PEGylated liposome loaded with doxorubicin) and Abraxane (���130 nm albumin-bound paclitaxel nano- particles), have been approved by the FDA, and have shown improved pharmacokinetics and reduced adverse effects compared to their parent drugs.5 However, currently approved nanomedicines provide modest survival benefits for patients,5,6 perhaps in part because of poor tumor penetration. Nanoparticle size is one crucial determinant of accumulation and penetration into tumor tissue.7 Nanoparticles with sub-100 nm sizes are optimal for the enhanced permeation and retention (EPR) effect for passive tumor targeting.8 However, physiological barriers, such as the dense interstitial matrix���a complex assembly of collagen, glycosaminoglycans, and proteoglycans���hinder the delivery of drugs throughout the entire tumor.9 For example, Doxil (���100 nm) is found trapped near the tumor vasculature.10 Although the small size (molecular weight = 544 Da) of doxorubicin released from Doxil allows rapid diffusion, doxorubicin cannot migrate far from the particles due to rapid uptake of doxorubicin by perivascular cells, which results in heterogeneous therapeutic effects.11 Deep penetration of nanoparticles in tumors is necessary to enhance their therapeutic effect.12 Another significant drawback of commercially available drug delivery NPs is that drugs are released at a predetermined rate irrespective of patient needs or changing physiological circumstances. A triggerable drug delivery system would allow repeated on-demand dosing that would be adaptable to the patients��� regimen and allow multiple dosages from a single administration.13 It might also help address the potential importance of timing on therapeutic effect (���chrono-admin- istration���) in the treatment of cancer,14 a concept that is receiving burgeoning recognition, for example, the periodicity of VEGF expression in breast cancer regulates tumor cancer vascular permeability.15 Another clinical example of the importance of timing is that periodic infusion of angiotensin II via the tail vein can enhance macromolecular delivery into tumors by overcoming the barrier of elevated interstitial fluid pressure within tumors no such increase of macromolecular uptake occurs either by an acute or chronic increase in blood pressure induced by angiotensin II.16 Furthermore, the permeability of many tumor models varies with time and in response to treatment, so that vascular pore sizes vary greatly, resulting in heterogeneous NP extravasation and drug delivery efficacy.5,17 On-demand drug release from NPs accumulated in tumors could allow in situ chrono-administration, potentially increasing drug retention in cancers, maximizing tumor killing and minimizing metastatic spread. Received: December 20, 2011 Article pubs.acs.org/JACS �� XXXX American Chemical Society A dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. XXXX, XXX, XXX���XXX

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- 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 ��-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- 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 ��max = 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 ��max 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 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). Journal of the American Chemical Society Article dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. XXXX, XXX, XXX���XXX B