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Light driven transformable optical agent with adaptive functions for boosting cancer surgery ...
Name: Light driven transformable optical agent with adaptive functions for boosting cancer surgery ...
Author: ji qi
Pages: 12
Year: 2017
Language: English
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J ablonski diagram helps clarify the basic principles of mole cular photophysics, which is closely correlated with the functionality and efficacy of molecular optical agents for cancer diagnosis (e.g.fluorescence and photoacoustic (PA) ima ging) and treatment (e.g. photodynamic therapy (PDT)) 1 3 .On the basis of Jablonski diagram, there are generally three energy dissipation pathways that probably occur after a chromophore absorbs light 4 6 : (1)fluorescence emission; (2) intersystem crossing to a triplet excited state, followed by generation of phosphorescence and/or reactive oxygen species (ROS) and (3) thermal deactivation via non radiation pathways. Among them, the absorbed energy for thermal deactivation is usually in direct proportion to the PA effect, as production of heat results in transient thermoelastic expansion and hence ultrasonic waves allowing for PA imaging 7 9 . Since the absorbed excitation energy isfixed in one chromophore, itsfluorescent and PA effects are always competitive 10 . It has been well established that quenching thefluorescence of a near infrared (NIR) light absorbing chro mophore is conducive to significantly boosting its PA signal 11,12 . Therefore, you cannot burn the candle at both ends; that is, utmostfluorescence and PA imaging, never both by far. However,fluorescence and PA imaging techniques have their own strengths and weaknesses, and more importantly, they have the characteristics of complementary advantages 13 . Fluorescence tech nique holds the advantage of excellent sensitivity but lacks of spatial resolution 14 . PA technique, on the other hand, offers centimetre scale deep imaging depth but suffers from low sensitivity 15 17 . Accordingly, the integration offluorescence and PA imaging modes decidedly enables precise diagnostic outcome by virtue of high sensitivity and imaging depth beyond the optical diffusion limit 18 21 . For this purpose, there have been a number of investigations to date reported that one material with NIR absorption could be simulta neously used for dual modalityfluorescence and PA imaging 20,21 . Nevertheless, this also implies that such material cannot try its best to do each optimally, as the photophysical working mechanisms of fluorescence and PA are nearly opposite to each other 4,22,23 . Therefore, development of an intelligent material with tunable photophysical properties, whose absorbed energy can be controlled to mostly concentrate on eitherfluorescence or PA channel as needed, is momentously desirable. To our knowledge, unfortunately, no such smart materials have been reported up to present. In this contribution, we report a smart function transformable nanoparticle (NP) based on a photo controllable molecule dithienylethene (DTE) (1 (4 (1,2,2 triphenylvinyl)phenyl)ethyli dene)malononitrile (TPECM) for considerable improvement of cancer surgery outcomes. DTE TPECM consisting of a DTE core and two surrounding TPECM units has closed ring and open ring isomers, reversibly switchable by external UV/visible light irradiation (Fig.1a). In the ring closing form, intramolecular energy transfer from TPECM to closed ring DTE and relatively planar geometric structure make thermal deactivation pathway dominate, leading to utmost absorbed energy focusing on PA imaging. In the ring opening form, however, both the molecular geometry and photophysical property totally change to make every effort to block the thermal deactivation, hence activating fluorescence emission and ROS production. It is found that the ring closing NPs generate noticeable PA signal output and pos sess good signal stabilities, which are superior to several com monly used PA contrast agents including semiconducting polymer nanoparticles (SPNs), methylene blue (MB) and indo cyanine green (ICG). Further surface modification of the NPs with a targeting moiety endows them specific tumour targeting ability. In vivo studies demonstrate that such intelligent NPs with controlled photophysical processes significantly boost the cancer surgery outcomes by harnessing the respective advantages of PA imaging,fluorescence imaging and PDT. This study thus providesa concept of function transformable optical agent with max imized effectiveness of each function, and verifies its great clinical potential in cancer diagnosis and treatment during surgery. Results Synthesis and characterization of photo controllable mole cules. Key synthesis steps of DTE TPECM are presented in Fig.1a. Suzuki cross coupling reaction was carried out between 1 (4 (1,2 diphenyl 2 (4 (4,4,5,5 tetramethyl 1,3,2 dioxaborolan 2 yl)phenyl) vinyl)phenyl)ethan 1 one (1)and3,3 (perfluorocyclopent 1 ene 1,2 diyl)bis(5 bromo 2 methylthiophene) (2) to produce the dike tone compound (3), which was further reacted with malononitrile to afford ROpen DTE TPECM as a yellow powder in a high yield. Detailed synthesis and characterization of the intermediates and final compound with nuclear magnetic resonance (NMR) and high resolution mass spectrum (HRMS) are shown in Supplementary Methods and Supplementary Figs1 17.ROpen DTE TPECMin THF shows intense absorption below 500nm (Supplementary Fig.18). Upon irradiation of such THF solution using 365nm light for 5min, ROpen DTE TPECM transforms to its ring closing isomer (RClosed DTE TPECM) as evidenced by the occurrence of a new absorption band from 520 to 800nm (Supplementary Fig.19a). Noteworthy, DTE TPECM molecule reversibly switches between the ring opening and ring closing states by external UV/ visible light exposure (Supplementary Figs19b,c). Thefluorescence properties of ROpen DTE TPECM and RClosed DTE TPECM were investigated. ROpen DTE TPECM exhibits typical aggregation induced emission (AIE) feature (Fig.1b, c): ROpen DTE TPECM in good solvent THF is non emissive due to the low frequency rotations of surrounding phenyl rings leading to rapid decay of the excited states; however, after formation of ROpen DTE TPECM aggregation by adding water (poor solvent) into THF solution, such intramolecular rotations are restricted by intermolecular steric hindrance, resulting in opening the radiative pathway 24 . In marked comparison with the brightfluorescence of ROpen DTE TPECM in aggregated state, there is no detectable photoluminescence (PL) emission of the ring closing isomer in both THF solution and aggregation form even extending the wavelength to 1200 nm (Supplementary Fig.20), because of the intramolecular energy transfer from thefluorescent TPECM to the non emissive ring closing DTE core 25,26 . Density functional theory gives the optimized geometric structures of RClosed DTE TPECM and ROpen DTE TPECM (for Cartesian coordinates see Supplementary Tables1,2). Owing to the closed ring, the two thiophene rings in RClosed DTE TPECM form a very planar conjugated structure. Compared with RClosed DTE TPECM, ROpen DTE TPECM has a more twisted 3D geometry with severely distorted structures of both the DTE core and TPECM arms thanks to the open ring (Fig.1a), which undoubtedly hinders the intermolecular interactions (e.g. stacking) when aggregated and thus significantly suppresses the non radiative decay pathways 27,28 . This hence explains why we choose TPECM as the arms to endow ROpen DTE TPECM with AIE effect, i.e. making every effort to block the thermal deactivation. Figure1d displays the X ray diffraction (XRD) profiles of the ring opening and ring closing isomers. Rather strong diffraction peaks (100, 200, 300 and 010) are observed in RClosed DTE TPECM, whereas there is only one weak peak (100) for ROpen DTE TPECM. This result verifies that the ring closing molecules with more planar structure induce much stronger intermolecular interactions 29 , agreeing well with the molecular geometry. Design principle of photo controllable molecules. The mole cular design rationale is summarized as follows. For RClosed DTE TPECM, the closed ring imparts a long wavelength ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8 2NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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absorption peak owing to the formation of low bandgap con jugated structure by the fused dithienylethene. Besides, TPECM is designed to contain tetraphenylethene (TPE) unit and electron deficient moiety (i.e. malononitrile), forming a donor acceptor (D A) structure, which enables efficient intramolecular charge transfer. Incorporation of such D Afluorophores thus causes a bathochromic shift, making the absorption maximum ofRClosed DTE TPECM well match the NIR pulsed laser excitation of PA imaging system. More importantly, in ring closing state, intramolecular energy transfer occurs that tremendously quen ches thefluorescence, and the relatively planar geometric struc ture of RClosed DTE TPECM also promotes intermolecular interactions. These vitally boost the non radiation pathways 30 . Accordingly, the energy balance of photophysics profoundly tilts O B O O SS BrBr F F F F FF SS F F F F FF OO SS F F F F FF CNNCNCCN 13 ROpen DTE TPECM 2 RClosed DTE TPECM UV light, ring closingVisible light, ring opening F F F F FF CNNCNCCN SS + Toluene 110 C, 5 h Side view No FL Side view 01020304050 60 70 80 90 95 a bc d 0 20406080100010203040 Water fraction (%) 450 500 550 600 650 700 750 95% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% PL intensity (au) Wavelength (nm) 0 5 10 15 20 25 300.05.0 10 4 1.0 10 4 1.5 10 4 (300)(010)(200) Intensity (au) 2 Theta (degree) RClosed DTE TPECM ROpen DTE TPECM(100)Pd(PPh 3 ) 4 , K 2 CO 3 THF/H 2 O 70 C, 24 h CH 2 (CN) 2 NH 4 OAc AcOH Colour: dark green Colour: light yellowStrong FLI/I 0 2.0 10 4 Fig. 1Synthesis, structure and property of photo controllable DTE TPECM molecules.aKey synthesis steps, photo controlled reversibility and optimized geometric structures of DTE TPECM molecules. Photographs of ROpen DTE TPECM and RClosed DTE TPECM powders in daylight and under UV light (365nm). FL:fluorescence.bPL spectra of ROpen DTE TPECM in THF/water mixture with various water fractions.cPlot ofI/I 0 versus water fraction.I 0 andIare the peak PL intensities of ROpen DTE TPECM (10 M) in pure THF and THF/water mixtures, respectively. Inset shows the photographs of ROpen DTE TPECM in THF/water mixtures with different water fractions taken under UV illumination.dXRD diagrams of ROpen DTE TPECM and RClosed DTE TPECM NATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8ARTICLE NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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to the thermal deactivation side when the ring is closed, bene fitting PA transition process (Fig.2). On the other hand, upon a simple visible light irradiation, the ring opens to yield ROpen DTE TPECM, which not only disrupts intramolecular energy transfer, but also transforms to a much more twisted 3D geometric structure favouring reduced inter molecular interactions. These tremendously inhibit the absorbed energy fromflowing to thermal deactivation, and thus adjust the energy balance of photophysics to incline to the opposite side (Fig.2), i.e.fluorescence emission and ROS generation (as ROpen DTE TPECM is not phosphorescent), allowing for fluorescence imaging and PDT. We hypothesized that, simply driven by external light, an overall majority of absorbed energy can be controlled to focus on either side of the balance, which makes our molecule exert its maximum potential for either PA imaging orfluorescence imaging plus PDT, and serve as a powerful optical agent for each different application. Preparation and characterization of function transformable NPs. To render the hydrophobic organic compounds with good in vivo biocompatibility, a nanoprecipitation method was adopted to formulate RClosed DTE TPECM or ROpen DTE TPECM using amphiphilic maleimide bearing lipid PEG 2000 as the dop ing matrix, yielding RClosed DTE TPECM doped or ROpen DTE TPECM doped lipid PEG 2000 NPs (in short, RClosed NPs and ROpen NPs, respectively). During the NP formation, the hydrophobic compounds and lipids entangle with each other and their formed aggregates act as the NP core, which is surrounded by the hydrophilic PEG outer layer that stabilizes the NPs (Fig.3a). Dynamic light scattering (DLS) and transmission elec tron microscopy (TEM) data show that both RClosed and ROpen NPs are spherical in shape with a similar average diameter of ~65 nm (Fig.3b, c). As presented in Fig.3d, RClosed NPs appear in blue green colour in aqueous solution, which possess an intense absorption peak centred at 650nm with a molar extinction coefficient of 4.4 10 4 M 1 cm 1 (Supplementary Fig.21). The PL spectra reveal that RClosed NPs are almost non fluorescent in water (Fig.3e and Supplementary Fig.20). Under continuous visible light (e.g. 610nm light) irradiation for 10min, theabsorption peak ranging from 520 to 800nm gradually decreases andfinally vanishes, which is accompanied by the solution colour changed to yellow and the emission peak at ~550nm significantly intensified, indicating the transformation from RClosed NPs to fluorescent ROpen NPs (Fig.3d, e). The ring closing and ring opening NPs can convert reversibly by alternating UV/visible light irradiation with negligible interference on the absorption, emission and PA properties during ten circles (Fig.3f and Sup plementary Fig.22), suggesting the highly reversible and bistable photochromism signature. It is also found that the RClosed NPs can effectively change to ROpen NPs even if the 610nm red light irradiation (0.3W cm 2 ) is through a 1cm thickness of chicken breast (Supplementary Fig.23). PA property of the ring closing NPs. The PA properties of RClosed NPs and ROpen NPs were studied by recording the PA intensity at different wavelengths from 680 to 840nm. RClosed NPs effectively generate PA signals under NIR pulsed laser irra diation with PA spectrum in good accordance with the absorp tion profile in the NIR region, whereas there is negligible PA signal detected from ROpen NPs (Fig.4a). A linear relationship is observed between PA intensity at 700nm and NP concentration based on RClosed DTE TPECM (Fig.4b). It is worthy to note that after exposure to 1.8 10 4 laser pulses at 700nm (1.5W cm 2 laser and 20Hz pulse repetition rate), nearly no loss of PA intensity is observed for RClosed NPs (Fig.4c), revealing the good photostability of RClosed NPs, which hardly convert to ROpen NPs under the PA imaging condition at 700nm. The PA signal and stability of RClosed NPs were then compared with several popularly used PA contrast agents, including SPNs, MB and ICG. The SPNs were prepared according to the literature by formulation of a semiconducting polymer poly(cyclopentadithiophene alt benzothiadiazole) using lipid PEG 2000 as the encapsulation matrix (Supplementary Fig.24) 4,31 . The absorption spectra of SPNs, MB and RClosed NPs in water suggest that they share similar maximal absorption wavelength (Supplementary Fig.25). Moreover, as the SPNs, MB and RClosed NPs are spectrally similar with PA maximum wavelength at about 680nm 31,32 , rational comparison is allowed S 1 S 1 S 0 S 0 T 1 T 1 ISC ISC A FL NR A FLNRO 2 ROSPA FL ROS FL ROSPAPhotophysical balancePA Ring closing Ring opening Fig. 2Illustration of the controllable photophysical processes. The energy balance of photophysics profoundly tilts to either side controlled by external UV/ visible light exposure. A: absorption, FL:fluorescence, NR: non radiation, ISC: intersystem crossing ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8 4NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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using a 680 nm pulsed laser. At the same condition, the PA intensityofRClosedNPsis~1.8 foldand~2.0 foldhigherthanthat of SPNs and MB, respectively (Fig.4d). Since the SPNs have been demonstrated as a high performing PA contrast agent even superior to single walled carbon nanotubes 31 and MB is also a commonly used molecule for PA imaging 16 , this comparison result illustrates that RClosed NPs can serve as an advanced PA molecular probe. High stability of probe signal against tumour endogenous reactive oxygen and nitrogen species (RONS) such as hypochlorite (ClO ), peroxynitrite (ONOO ) and hydroxyl radical ( OH) is an important prerequisite for accurate cancer diagnosis 33 35 .As depicted in Fig.4e, in the presence of various RONS, the absorption spectra of RClosed NPs and SPNs hardly change, indicating that they are RONS inert. In sharp contrast, ICG shows the worst performance in resisting RONS with absorption band rapidly decreasing after addition of each RONS. Moreover, MB is not stable against ONOO , regardless of the good resistance towards ClO and OH. As SPNs are advantageous due to their excellent RONS resistance 36,37 , the result proves that RClosed NPs are promising for precise in vivo PA cancer imaging. Fluorescence property and ROS generation of the ring opening NPs. PL excitation mapping was performed on ROpen NPs, displaying excitation and emission peaked at ~410 and ~550nm, respectively (Fig.4f). The NIR absorption completely disappears after RClosed NPs transform to ROpen NPs. Thefluorescence quantum yield ( F ) and lifetime ( ) of ROpen NPs are measured to be 23.8% and 1.45 ns (Fig.4g), respectively, while RClosed NPs show no detectable F and . The ROpen NPs are tolerant to ClO , ONOO and OH, as indicative of the unchanged emission spectrum in the presence of each RONS (Supplementary Fig.26). The ROpen NPs also exhibit similar anti photobleaching capacity to commercial QD585 (Supplementary Fig.27), which is well known for its ultrahigh photobleaching threshold 38 . For photosensitizers, the absorbed energy can transfer to the triplet excited state via intersystem crossing, followed by generationof ROS that are singlet oxygen in most cases 5,39 41 . The abilities of the RClosed and ROpen NPs to produce ROS upon light excitation were examined utilizing 2 ,7 dichlorodihydrofluorescein diacetate (DCF DA) as the ROS indicator 42 . As depicted in Fig.4h, RClosed NPs hardly generate ROS upon excitation at either 365 nm or 610 nm. On the contrary, efficient ROS generation is observed for ROpen NPs upon excitation at 365nm by monitoring the fluorescence activation due to the oxidation reaction between ROS and non emissive DCF DA to yieldfluorescent dichlorofluorescein (DCF) 42 . Additionally, after conversion of RClosed NPs to ROpen NPs via exposure to 610nm light, the converted ROpen NPs can efficiently produce ROS under subsequent 365 nm light irradiation. It is also validated that ROpen NPs are capable of effectively generating ROS upon exposure to white light (Fig.4i), as white light (400 700 nm) irradiation has been widely accepted for in vivo PDT 43 . NP surface modification with a targeting ligand. It has been reported that the peptide with a sequence of YSAYPDSVPMMS (named YSA in short) is able to selectively and tightly bind to EphA2 protein, which is a transmembrane receptor tyrosine kinase overexpressed in many cancer cells as well as tumour blood vessels including 4T1 mammary adenocarcinoma 44,45 . Therefore, YSA peptide was employed as a targeting ligand to modify our function transformable NPs in order to endow them with active tumour targeting ability. We synthesized CYSAYPDSVPMMS peptide (Fig.5a) with a terminal thiol group in cysteine (C) via standard 9 fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS), which was characterized by LC MS and HRMS (Supplementary Figs28,29, and details see Supplementary Methods). Our function transformable NPs were then modified with CYSAYPDSVPMMS peptide through the coupling reaction between the thiol group of peptide and the maleimide group of PEG on the NPs, affording YSA conjugated NPs (namely RClosed YSA NPs or ROpen YSA NPs; Fig.5b). It is calculated that there are ~3700 YSA peptides on average 024680 s 5 s0 s def 0 cb RClosed NPs ROpen NPs ROS FLPA RClosed NP a Visible light UV light ROpen NP Intensity (counts) 100 80 60 40 20 Diameter (nm) 16060 80 100 120 1400100 80 60 40 20 Diameter (nm) 16060 80 100 120 140 Intensity (counts) Absorption (au) 0.3 0.2 0.1 0.0 Wavelength (nm) 800550 600 650 700 75015 s 30 s 45 s 60 s 75 s 90 s 120 s 150 s 180 s 210 s 300 s 450 s 600 s0 s 5 s 15 s 30 s 45 s 60 s 75 s 90 s 120 s 150 s 180 s 210 s 300 s 450 s 600 s600 s PL intensity (au) 700450 500 550 600 650 Wavelength (nm) Absorption (au) 0.4 0.3 0.2 0.1 0.0 Number of cycle 10Lipid PEG; RClosed DTE TPECM; ROpen DTE TPECM Fig. 3Preparation and characterization of the photo controllable NPs.aSchematic of RClosed NPs and ROpen NPs.b,cDLS profilesandTEMimagesofb RClosed NPs andcROpen NPs. Scale bars, 100nm for TEM images.dAbsorption andePL spectra of RClosed NPs under visible light (610nm, 0.3 Wcm 2 ) irradiation for different time as indicated. Photographs indindicate the aqueous solutions of RClosed NPs before and after 610nm light exposure for 600s.f The absorption intensity at 650nm of the NPs during ten circles of visible (610nm, 0.3Wcm 2 )/UV light (365nm, 0.1 Wcm 2 ) irradiation processes NATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8ARTICLE NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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conjugated on each NP (Supplementary Methods Equation 3). The mean size of YSA conjugated NPs is ~68 nm determined by DLS, which is similar to that of the NPs without YSA. It is also demonstrated that the YSA modification does not influence any of the NP properties in terms of PA andfluorescence properties, ROS generation capacity and reversible photochromism. In vitro cellular study was then carried out with 4T1 murine breast cancer cells. Using hepatic L02 normal cells as a control, the western blot study reveals that EphA2 is predominantly expressed in 4T1 cancer cells (Fig.5c and Supplementary Fig.30). It is demonstrated that the YSA conjugation significantly improves the NP internalization by 4T1 cancer cells because of the strong interaction between YSA and EphA2 receptor overexpressed on the cancer cell membrane 44,45 , and that the RClosed YSA NPs can be facilely transformed to the ROpen YSANPs inside the 4T1 cells, triggered by 610nm light irradiation (Supplementary Figs31and32). Additionally, 610nm light (0.3 Wcm 2 ) irradiation itself does not cause the photothermal effect (Supplementary Fig.33) and the treatment of'RClosed YSA NPs+610nm light irradiation (0.3 W cm 2 , 5min)'results in negligible cytotoxicity (Fig.5d), implying the good biocompat ibility of the NPs and the harmless of red light irradiation under the experimental condition. It is also found that the converted ROpen YSA NPs within 4T1 cancer cells have good ROS generation ability (Supplementary Fig.34) and more effective in vitro PDT efficacy than the converted ROpen NPs (Fig.5e). In vivo pharmacokinetics and biodistribution. After we demonstrated that both the RClosed YSA and ROpen YSA NPs 0.00123 PA amplitude (au) Number of pulses 0 100 200 300 0102030405060 Irradiation time (s) ROpen NPs white light DCF DA alone RClosed NPs SPNs Pristine MB ICG 051015 PA intensity (au) 0 100 200300048121620 RClosed NPs 365 nm RClosed NPs 610 nm RClosed NPs (i) 610 nm (ii) 365 nm ROpen NPs 365 nm ROpen NPs 610 nm Irradiation time (s) ig 650 700 750 800 8501234 PA intensity (au) Wavelength (nm) RClosed NPs ROpen NPs bca h edf 390400410420430440 High 700650600550500 Emission wavelength (nm) Excitation wavelength (nm) 450Low 0 2040608010004080120160200 RClosed NPs ROpen NPs PA intensity (au) Concentration ( M) 5 1015202530 Intensity (au) Time (ns)6.0 10 3 1.2 10 4 1.8 10 4 RClosed NPs ROpen NPs SPNs MB ICG PBS A/A 0 .OHONOO ClO 10 4 10 3 10 2 10 1 10 0 I/I 0 I/I 0 Fig. 4In vitro PA,fluorescence and ROS generation of the NPs.aPA spectra of RClosed and ROpen NPs.bPA intensities of RClosed and ROpen NPs at 700nm as a function of molar concentration based on DTE TPECM molecules. Error bars, mean s.d. (n=3) fora,b.cPA amplitudes of RClosed NPs as a function of number of laser pulses (1.8 10 4 pulses; 1.5Wcm 2 laser and 20Hz pulse repetition rate).dPA intensities excited with 680nm pulsed laser of various agents at the same molar concentration (100 M) based on MB, ICG, DTE TPECM molecules and the repeat unit of SP.ePlot ofA/A 0 versus different RONS.AandA 0 are the absorption intensity at 680nm of RClosed NPs, SPNs, MB and ICG in the presence and absence of RONS (400 M), respectively. Error bars, mean s.d. (n=3) for (d,e).fPL excitation mapping andgfluorescence decay curve of ROpen NPs.hPlot ofI/I 0 versus light irradiation time. The aqueous solution of RClosed NPs or ROpen NPs (10 M based on DTE TPECM) was exposed to 610nm red light (0.3Wcm 2 ) and/ or 365nm UV light (0.1Wcm 2 ).iPlot ofI/I 0 versus white light (0.25Wcm 2 ) irradiation time of ROpen NPs (10 M based on ROpen DTE TPECM) in aqueous solution.I 0 andIare the PL intensity of DCF at 525nm before and after light irradiation at designated time intervals in bothh,i ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8 6NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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can be safely utilized for in vivo application through a series of blood chemistry examinations and histological analyses of impor tant normal organs (Supplementary Figs35 37), in vivo pharma cokinetics of the function transformable NPs was investigated. As radiolabelling is a routine and reliable method to trace administered species in in vivo pharmacokinetic studies 46 ,RClosed YSANPs were radiolabelled with a radioactive nuclide, iodine 125 ( 125 I), which was reacted with the tyrosine (Y) residues of YSA peptide. The radiochemical purity of 125 I labelled RClosed YSA NPs is higher than 99%, which does not change upon keeping the NPs in saline for 3 days, revealing the high radiolabelling stability. As RClosed YSA NPs serve as the staple imaging probe/therapeutic agent in the next cancer surgery study, their pharmacokinetics was evaluated in healthy rats benefitting from 125 I labelling. After 125 I labelled RClosed YSA NPs were intravenously injected into the rats, the blood samples were collected at designatedtime intervals and counted for 125 I radioactivity with a gamma counter. Figure5f displays the blood circulation behaviour of 125 I labelled RClosed YSA NPs. The circulation half life, the volume of distribution and the blood clearance of the RClosed YSA NPs are determined to be 6.21 0.39h, 143.03 12.12mLkg 1 and 24.47 2.38mLkg 1 h 1 , respectively (see Supplementary Table3 for complete pharmacokinetic data). The biodistribution of 125 I labelled RClosed YSA NPs in tumour bearing mice was investigated as well. The xenograft 4T1 tumour bearing mouse model was employed, which was established by subcutaneous inoculation of 4T1 cancer cells into the mouse right axillary space. After 125 I labelled RClosed YSA NPs were administrated into 4T1 tumour bearing mice through the tail vein, the time dependent biodistributions of the NPs in blood, tumour and various major organs of mice were quantitatively analysed by gamma scintillation counting (Fig.5g). 04816400306090120 Converted ROpen YSA NPs Converted ROpen NPs Cell viability (%) 0481640800306090120 Cell viability (%) 0 4 8 12 16 20 240102030 Time (h) Blood Skin Muscle Intestine Heart Lung Liver Kidney Spleen Stomach Bone Tumour 8 h 24 h 48 h 72 h 0.083 h 0.5 h 1 h 2 h 4 h 030 1050 40 20 gf CYSAYPDSVPMMS (YSA) peptide NH HN NH HN N HN NH HN N HN NH HN OH O OO O O O O O O O O CH 3 OH O OH CH 3 O OH OH OH OH S CH 3 S CH 3 O SH H 2 N da : 125 IHS NO O SH + RClosed NP bc L02 Normal cells4T1 Cancer cells (ii) (i) e SPPS * * (i): EphA2 (107 kDa) (ii): GAPDH (36 kDa) H 3 C YSA conjugated RClosed NP (Rclosed YSA NP) Concentration ( M) 125 I labeled RClosed YSA NP %ID g 1 %ID g 1 Concentration ( M) Fig. 5Targeting modification, cytotoxicity and in vivo pharmacokinetics.aChemical structure of CYSAYPDSVPMMS peptide. SPPS solid phase peptide synthesis.bSchematic of the preparation of a RClosed YSA NP.cWestern blot analyses of EphA2 in L02 cells and 4T1 cancer cells.dCell viability of RClosed YSA NP incubated 4T1 cancer cells after 610nm red light (0.3Wcm 2 ) irradiation for 5min. Error bars, mean s.d. (n=4).eCell viabilities of the converted ROpen YSA NP loaded and converted ROpen NP loaded 4T1 cancer cells under white light irradiation (0.25Wcm 2 , 4min). Error bars, mean s.d. (n=4). *P<0.05, unpaired Student'st test (two tailed). The cells were incubated with RClosed YSA and RClosed NPs, respectively, followed by exposure to 610nm red light (0.3Wcm 2 ) for 5min, to obtain the converted ring opening NP loaded cells. Indande, the concentration is based on DTE TPECM.fPharmacokinetics study of 125 I labelled RClosed YSA NPs analysed by scintillation count of 125 I radioactivity in blood. Error bars, mean s.d. (n=6 rats). Inset displays the schematic of an 125 I labelled RClosed YSA NP.gBiodistribution of 125 I labelled RClosed YSA NPs in various tissues of 4T1 tumour bearing mice at different time points post intravenous injection. Error bars, mean s.d. (n=6 mice for each time point) NATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8ARTICLE NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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It is obvious that the RClosed YSA NPs rapidly leave the bloodstream and enter most organs, and the levels of the NPs in all of the tissues significantly decrease after 8h. Due to the reticuloendothelial system and mononuclear phagocyte system uptake 47 , high accumulations of the NPs in liver, spleen and bone marrow are found. Importantly, thanks to both the active (YSA EphA2 interaction) and passive (the enhanced perme ability and retention (EPR) effect of nanomaterials) 48 tumour targeting capabilities, the RClosed YSA NPs can be largely enriched in tumour tissue with maximum tumour uptake of ~7.3% ID g 1 occurring at 4h post injection. Improvement of cancer surgery outcomes. We next investigated whether the function transformable NPs could improve cancer surgery outcomes. As PA technique permits imaging thatsurpasses the limit of optical diffusion 15,16 , compared with fluorescence imaging, PA imaging could offer relatively deeper information on the tumours in vivo before surgery. A commercial small animal opt acoustic tomography system (MOST) was used to study the utility of RClosed YSA NPs in in vivo PA imaging of tumours. As shown in Fig.6a, before NP administration (0 h), there is weak PA signal at 700nm in the tumours of living mice, probably attributed to the intrinsic background by oxyhemoglo bin and deoxyhemoglobin 4 . Subsequently, the RClosed YSA NPs (100 L, 800 M based on RClosed DTE TPECM) were injected into one group of 4T1 tumour bearing mice via the tail vein. As a control, the same amount of RClosed NPs without YSA mod ification was intravenously administrated into the other group of tumour bearing mice. In vivo PA imaging was then conducted after injections. For the mice in both two groups, the PA signals in tumours significantly elevate and reach the maximum at 4h, c BrightfieldFL channel H&EH&E FL channel dfea 0 h 4 h 24 h Max. Min. b 0 5 10 15 20 25100200300400 PA intensity (au) Time (h) RClosed NPs RClosed YSA NPs * * * * RClosed YSA NPsRClosed NPs Pre surgery Post surgery Post surgery; IT: 0 min Post surgery; IT: 5 min Post surgery; IT: 0 min Post surgery; IT: 5 min Fig. 6In vivo preoperative PA imaging and intraoperativefluorescence imaging.aRepresentative time dependent PA images of subcutaneous tumours from mice intravenously injected with RClosed YSA and RClosed NPs (800 M based on RClosed DTE TPECM, 100 L), respectively. Scale bars, 2mm.b Plot of PA intensity at 700nm in tumour versus time post injection of RClosed YSA or RClosed NPs. Error bars, mean s.d. (n=3 mice per group). *P< 0.05, in comparison between RClosed YSA and RClosed NPs using unpaired Student'st test (two tailed).cRepresentative brightfield images of RClosed YSA NP treated tumour bearing mice before and after surgery as well as representativefluorescence images of mice with complete surgical resection of tumours, followed by 610nm red light (0.3Wcm 2 ) irradiation at the operative incision site for 5 min. Scale bars, 3mm. FLfluorescence, IT irradiation time.dH&E stained tissues at the operative incision site incindicate no residual tumours left behind. Scale bar, 1 mm.eRepresentativefluorescence images of RClosed YSA NP treated mice with residual tumours post surgery. The operative incision site was irradiated by 610nm red light (0.3Wcm 2 ) for 5min. The red dashed circles incandeindicate the tumour/operative incision site. The red arrow shows the residual tumours with a diameter below 1 mm. Scale bars, 3mm.f, H&E stained tissues at the operative incision site ineconfirm the existence of residual tumours. Scale bar, 0.5mm ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8 8NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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which then gradually decrease as the time elapses (Fig.6a, b), agreeing well with the biodistribution data. Noteworthy, the average PA signal in RClosed YSA NP treated tumours is sta tistically higher than that in RClosed NP treated tumours at each tested time point (for example, ~1.8 times higher at 4h post injection) (Fig.6b). With the information provided by preoperative PA imaging, surgery can be performed to excise the tumours in vivo. In the clinic, one of the most challenging issues during cancer surgery is to quickly assess whether all the tumour masses have been removed without any residual tumours left behind 49 . Addressing this challenge requires a highly sensitive imaging modality in combination with a highly effective contrast agent. In this regard, fluorescence imaging is a promising candidate, since it is sensitive, fast, real time and instrument portable 50 . In our experiment, after tumour resection with the aid of PA imaging using RClosed YSA NPs, 610nm light was immediately irra diated at the operative incision site for 5min. Interestingly, if the tumours are totally removed by the surgeon, which is confirmed by hematoxylin and eosin (H&E) histological analyses, no fluorescent signal can be detected at/around the incision site (Fig.6c, d). Nevertheless, if there are residual tumours left behind post resection, thefluorescent signal gradually turns on at/around the incision site within 5min irradiation duration (Fig.6e), which arises from the RClosed YSA NPs in the residual tumours rapidly transforming tofluorescent ROpen YSA NPs. The existence of residual tumours is verified by H&E staining (Fig.6f). As a control, when residual tumours of saline treated mice are irradiated with 610nm light for 5min, no detectablefluorescent signal can be seen (Supplementary Fig.38). This confirms that the light upfluorescence from residual tumours indeed originates from the transformed ROpen YSA NPs. It is important to note that the ratio of averagefluorescence intensity from the residual tumours to that from surrounding normal tissues is ~7.1, which outperforms the Rose criterion and is higher than the reported values of ICG and MB influorescence imaging guided surgery 13,44 . Thanks to the large signal to background ratio, submillimeter tumours can also be clearly delineated by the light upfluorescence of our NPs, indicated by the red arrow in Fig.6e. For the mice with transformed ROpen YSA NPs indicating negligible residual tumours, 18 of 20 mice were cured without any in situ tumour recurrences and survived 2 months. On the other hand, all the 20 mice with residual tumours visualized by converted ROpen YSA NPsfluorescence experienced fast growth of residual tumours and died within 2 month monitoring duration. Thereby, our function transformable NPs can improve cancer surgery outcomes by preoperative cancer diagnosis via PA imaging together with intraoperativefluorescent visualization of residual tumours in a sensitive, fast and real time manner, significantly reducing the risk of in situ tumour recurrence. In many clinical cases, complete tumour resection is impossible or not suggested. Aiming for this, surgical debulking of tumours that refers to removal of most of a surgically incurable malignant tumour has been advocated for many cancers such as ovarian carcinoma, lymphoma, sarcoma and neoplasms of central nervous system, which is very common in the clinic 51 . One major purpose of debulking is to improve the quality of life and extend survival despite of not curing the cancer thoroughly. Generally, subsequent treatment after debulking surgery must be carried out to control the tumours left behind 52 . As the ring opening NPs serve as an efficient photosensitizer, we wonder whether the PDT of ring opening NPs within residual tumours post debulking surgery can significantly impede the residual tumour growth and thus prolong the patients'lifetimes. As such, subcutaneous 4T1 xenograft tumour bearing mice were randomly assigned tofive groups, named'debulking surgery (DS) alone','DS+Light','DS+YSA NPs','DS+NPs+Light'and'DS+ YSA NPs+Light', respectively. It is worthy pointing out that the 4T1 cancer cells in this study express luciferase, allowing for tracking the tumours via bioluminescence imaging (for details see the Methods section). On day 0, RClosed YSA NPs were intravenously injected into the mice in both'DS+YSA NPs' and'DS+YSA NPs+Light'cohorts. Moreover, RClosed NPs were intravenously administrated into the mice in'DS+NPs+ Light'group. At 4h post injection, the tumours of all the mice in five groups were debulked. The mice sharing similar residual tumours in terms of size and activity determined by biolumines cence imaging were selected with each group containing ten mice. For the mice in'DS+Light','DS+YSA NPs','DS+NPs+ Light'and'DS+ YSA NPs+Light'groups, after debulking, 610 nm light irradiation (0.3W cm 2 ) was performed at the incision site for 5min to convert ring closing NPs to ring opening NPs in the residual tumours. This was followed by white light irradiation for another 5min on residual tumours from mice in'DS+Light', 'DS+NPs+Light'and'DS+YSA NPs+Light'groups to make ring opening NPs generate ROS for PDT. After various treatments on day 0, the size and activity of residual tumours from mice in allfive groups were monitored for 15 days by bioluminescence imaging. As shown in Fig.7a, b, as compared to DS alone causing fast growth of residual tumours, DS followed by PDT with no matter RClosed YSA NPs or RClosed NPs gives rise to good efficacy on suppression of residual tumours, as evidenced by the growth stoppage of residual tumours in both'DS+YSA NPs+Light'and'DS+NPs+Light'groups. It is worthy to note that'DS+YSA NPs+Light'is the only treatment that achieves smaller average tumour size on day 15 than that on day 0, leading to better antitumor efficacy as compared to'DS+NPs+Light'. As controls, the treatments of'DS+Light'and'DS+YSA NPs' fail to be efficacious on residual tumour inhibition (Fig.7band Supplementary Fig.39), demonstrating that the impressive antitumour activity indeed roots in the PDT of YSA conjugated NPs post DS. Furthermore, 9 of 10 mice in' DS+YSA NPs+Light' cohort and 7 of 10 mice in'DS+NPs+Light'group could survive 40 days, whereas the mice in the other three groups all died within 40 day study duration (Fig.7c). These results manifest that our strategy using the function transformable NPs is also efficacious to improve DS outcomes, effectively prolonging the lifetimes of tumour bearing mice after DS. Discussion RClosed DTE TPECM is rationally designed to make every effort to concentrate utmost absorbed energy on the pathway of thermal deactivation for PA imaging, in terms of intramole cular energy transfer to quenchfluorescence, and relatively planar geometric structure to promote intermolecular interac tions (Fig.1a, d). These enable RClosed NPs neitherfluoresce nor generate any ROS, but generate brighter PA signal than the reported high performing SPNs and MB (Fig.4d) 4,31 . Upon simple irradiation by visible light, the DTE ring opens to afford ROpen DTE TPECM, which however is designed to make every effort to restrain the thermal deactivation pathway, favouring absorbed energyflow to the other two energy dissipa tion pathways, i.e.fluorescence emission and intersystem crossing to triplet excited state to generate ROS. In addition to open ring cancelling intramolecular energy transfer, AIE active TPECM is employed to endow the molecule with a clawed 3D geometry for depressing intermolecular interactions (Fig.1a, d). These sig nificantly block the non radiative decay, leading to ROpen NPs generating negligible PA signal (Fig.4a, b) and thus becoming an effectivefluorescent probe (Fig.4f, g) and photosensitizer (Fig.4h, i). NATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8ARTICLE NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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Surgery is one of the most adapted strategies to treat solid tumours 52 . For clinical cancer surgery, preoperative imaging and intraoperative imaging call for different imaging techniques 50 .The RClosed YSA NPs after intravenous administration are capable of delineating tumours via PA imaging before surgery, attributable to their high PA brightness as well as their active (YSA EphA2 interaction) and passive (EPR effect) tumour targeting capabilities (Fig.6a, b). If there are residue tumours post resection, the trans formedfluorescent ROpen YSA NPs can sensitively visualize them via simple exposure of operative incision site to 610nm light for 5 min, which give a high tumour to normal tissue ratio of ~7.1 and even permit clear detection of residual tumours below 1mm in diameter (Fig.6e). As a consequence, our light driven function transformable NPs show good performances in both preoperative PA imaging and intraoperativefluorescence imaging, significantly reducing the risk of in situ tumour recurrence. In cases of DS that complete tumour resection is impossible or not suggested, the PDT of transformed ROpen YSA NPs within residual tumours post DS are efficacious on suppression of residual tumour growth and prolongation of the lifetimes oftumour bearing mice (Fig.7). Such dramatic PDT efficacy not only lies in the effective ROS production of ROpen YSA NPs, but more importantly, attributes to surgery helping overcome two major limitations of PDT, i.e. limited tissue penetration depth of excitation light and insufficient oxygen within big tumours 53,54 . Therefore, this study demonstrates that PDT is quite suitable and efficacious for treatment of residual tumours after DS and our smart NPs remarkably promote the DS outcomes. In summary, we have developed a function transformable NP that can serve as powerful PA contrast agent,fluorescent probe and photosensitizer as needed, simply triggered by external light, which give excellent performance in boosting the cancer surgery outcomes. Such smart NPs with controlled photophysical prop erties show unique merits over all other existing optical agents in terms of the combined advantages of simple but'one for all' system, on demand function tunability and utmost effectiveness of each function. This study therefore creates a class of optical agents with absorbed energy convertible and function transformable signatures for advanced biomedical application at a comprehensive level not achievable by currently reported optical 0369121518 10 5 10 6 Time (day) DS + YSA NPs + Light DS + NPs + Light DS + YSA NPs DS + Light DS aloneDay 0 Day 2 Day 7 Day 15 DS + light DS alone a ** DS + NPs + lightDS + YSA NPs + light 5 b c 0 102030400.000.250.500.751.00 Survival rate Time (day) DS alone DS + Light DS + YSA NPs DS + NPs + Light DS + YSA NPs + LightPost surgery 10 1520 25 30 10 6 (p/s/cm 2 /sr)10 7 Average bioluminescence intensity (photons/s/cm 2 /sr) Fig. 7In vivo PDT of residual tumours after debulking surgery (DS).aTime dependent bioluminescence imaging of residual tumours from mice in different groups. The tumours were debulked on day 0. The 4T1 cancer cells express luciferase, permitting bioluminescence imaging. The black arrows indicate the residual tumours.bQuantitative analysis of bioluminescence intensities of residual tumours from mice with various treatments as indicated. Error bars, mean s.d. (n=10 mice per group). **P<0.01, one way ANOVA.cSurvival curves for different groups (n=10 mice per group) ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/s41467 018 04222 8 10NATURE COMMUNICATIONS| (2018) 9:1848 |DOI: 10.1038/s41467 018 04222 8|

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