In vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics

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1 Reeived 27 May 2015 Aepted 7 De 2016 Published 20 Jan 2016 DOI: /nomms10432 OPE In vivo ovalent ross-linking of photon-onverted rare-earth nanostrutures for tumour loalization and theranostis Xiangzhao Ai 1, Chris Jun Hui Ho 2, Junxin Aw 1, Amalina Binte Ebrahim Attia 2, Jing Mu 1, Yu Wang 3, Xiaoyong Wang 4, Yong Wang 5, Xiaogang Liu 3,6, Huabing Chen 5, Mingyuan Gao 5, Xiaoyuan Chen 4, Edwin K.L. Yeow 1, Gang Liu 4, Malini Olivo 2 & Bengang Xing 1,6 The development of preision nanomediines to diret nanostruture-based reagents into tumour-targeted areas remains a ritial hallenge in linis. Chemial reation-mediated loalization in response to tumour environmental perturbations offers promising opportunities for rational design of effetive nano-theranostis. Here, we present a unique miroenvironment-sensitive strategy for loalization of peptide-premodified uponversion nanorystals (UCs) within tumour areas. Upon tumour-speifi athepsin protease reations, the leavage of peptides indues ovalent ross-linking between the exposed ysteine and 2-yanobenzothiazole on neighbouring partiles, thus triggering the aumulation of UCs into tumour site. uh enzyme-triggered ross-linking of UCs leads to enhaned uponversion emission upon 808 nm laser irradiation, and in turn amplifies the singlet oxygen generation from the photosensitizers attahed on UCs. Importantly, this design enables remarkable tumour inhibition through either intratumoral UCs injetion or intravenous injetion of nanopartiles modified with the targeting ligand. Our strategy may provide a multimodality solution for effetive moleular sensing and site-speifi tumour treatment. 1 Division of Chemistry and Biologial Chemistry, hool of Physial and Mathematial ienes, anyang Tehnologial University, ingapore, ingapore. 2 ingapore Bioimaging Consortium, Ageny for iene Tehnology and Researh (A*TAR), ingapore, ingapore. 3 Department of Chemistry, ational University of ingapore, ingapore, ingapore. 4 tate Key Laboratory of Moleular Vainology and Moleular Diagnostis, Center for Moleular Imaging and Translational Mediine, hool of Publi Health, Xiamen University, Xiamen, China. 5 hool of Radiation Mediine and Protetion, oohow University, uzhou, China. 6 Institute of Materials Researh and Engineering, A*TAR (Ageny for iene, Tehnology and Researh), ingapore, ingapore. Correspondene and requests for materials should be addressed to G.L. ( gangliu.mitm@xmu.edu.n) or to M.O. ( malini_olivo@sbi.a-star.edu.sg) or to B.X. ( Bengang@ntu.edu.sg). ATURE COMMUICATIO 7:10432 DOI: /nomms

2 ATURE COMMUICATIO DOI: /nomms10432 Currently, therapeuti and diagnosti tehniques based on supramoleular assemblies and funtional nanomaterials have been extensively reognized as promising nanomediine platforms for the battle against many urgent health onerns inluding aner, ardiovasular and neurodegenerative diseases as well as other life-threatening illnesses 1 3. The remarkable biomedial appliation of nanomaterials ould be mainly attributed to their unique photo-physial properties, high surfae area and multivalent binding ability 4,5. Despite the leap forward in the ontinuous breakthroughs in biomedial researh, ritial hallenge still remains in designing targeted nanoplatforms that are apable of seletively loalizing at the speifi diseases in partiular tumour sites for early-stage diagnosis and effetive treatment 6 8. One emerging strategy to ahieve high targeting seletivity is to onjugate the nanomaterials with affinity ligands inluding small organi moieties or bioative moleules that an bind to reeptors in the tumour ells However, varying expression levels of the reeptors, omplex and dynami physiologial ell environments may potentially pose the issue of nonspeifi reognition for this ligand-mediated tumour affinity. Therefore, more speifi targeting approahes are demanded that do not solely rely on reeptors to differentiate tumour and normal ells 11,12. Indeed, some bioorthogonal reations provide feasibility to loate funtional nanostrutures into tumour ells mostly through their eletrostati or ovalent binding to biomoleules in living system evertheless, the effetive bioorthogonal funtionalities that an seletively respond to the dynami proesses of native environment are still ongoing hallenges for in vivo appliations Hene, different approahes that enable sensitive reognition of dynami tumour miroenvironment, and more importantly, an further trigger the tumour-speifi loalization of theranosti nanomaterials in vivo are highly desirable, and extensive studies still need to be further investigated. Reently, rare-earth doped uponversion nanorystals (UCs) have been widely demonstrated for use in biomedial appliations. In general, UC partiles offer deep tissue penetration apability for enhaned bioimaging and better tumour treatment arising from their unique non-linear photon uponverting proess upon light irradiation at near-infrared (IR) window As with the majority of nanomaterials for theranosti tumour studies, the effetive targeting of uponversion materials mainly relies on reeptor-mediated interations, and the speifi ellular loalization of UC nanostrutures at the tumour site upon the sensitive response to miroenvironment stimulation have not been fully solved Moreover, despite the great potential of UCs in meeting biomedial demands in vitro and in vivo, mostofthe onventionally used UCs employ long-wavelength laser irradiation at 980 nm. This often leads to an inevitable overheating effet beause of signifiant water absorption 35,36. To this end, the development of new strategies to seletively diret UCs into targeted tumour areas, while notably minimizing undesired side effets is highly essential. Unfortunately, suh relevant studies are still limited so far. In this work, a tumour miroenvironment-sensitive UCs platform has been designed and prepared to perform in vivo ovalent loalization of partiles at the tumour site. Different from the proess involving nonspeifi tumour targeting, suh unique platform an respond to tumour-speifi enzyme and undergo ross-linking reation, whih thus enables the seletive tumour aumulation. More signifiantly, ompared with the partiles that annot undergo ross-linking reation, the enzymetriggered ovalent ross-linking of UCs possess an enhaned light uponverting emission when illuminated at 808 nm. uh enhanement an effetively amplify the prodution of reative singlet oxygen (for example, 1 O 2 ) from the photosensitizers loaded on the partile surfae, whih therefore represents promising nanomediine for improved photodynami tumour treatment (PDT) as well as non-invasive fluoresene and photoaousti imaging in vitro and in vivo. Results Enzyme-triggered ovalent ross-linking of UC in vitro. Figure 1 illustrates the rational design of our enzyme-responsive ross-linking of rare-earth UCs () for tumour loalization and targeted therapeutis. Generally, the ore-shell UCs were synthesized aording to an established thermal deomposition method 26. In order to minimize undesired overheating effet typially assoiated with 980 nm laser irradiation, we onstruted lanthanide platform doped with d 3 þ ions for improved pumping effiieny at 808 nm. These d 3 þ -doped nanorystals were further oated with polyaryli aid (PAA) and polyethylenimine (PEI) to improve bioompatibility and to failitate subsequent surfae modifiation (upplementary Fig. 1). Transmission eletron mirosopy and spetrosopi analysis revealed a ubi morphology of the partiles with a narrow size distribution of 40±2 nm. Both ore-shell UCs and polymeroated UCs exhibited similar uponverted luminesenes with visible emission at 545 and 655 nm upon exitation at 808 nm (upplementary Fig. 2). To ensure the theranosti effiay, an effetive photosensitizer, hlorin-e6 (Ce6), with favourable optial properties was hosen and oupled to the PEI/PAA@UCs. In order to ahieve seletive tumour loalization, an enzyme-responsive peptide, A-FKC(tBu)AC(H)-CBT (upplementary Fig. 3) ontaining a side-proteted ysteine and 2-yanobenzothiazole (CBT) was designed for speifi reation with athepsin B (CtsB), one important lysosomal ysteine protease overexpressed in various malignant tumours to proess intraellular protein degradation and regulate aner pathology 37. The seleted sequene was further onneted to Ce6-PEI/PAA@UCs through the reation of free thiol in terminal ysteine and short maleimide oligo (ethyl glyol; dpeg 2 ) linker on the partile surfae. The suessful formation of peptide and Ce6-modified UCs () onjugate was onfirmed by spetrosopi haraterization (upplementary Fig. 4). The optimal amount of peptide on UCs was finally determined to be 11.4 nm mg 1, and there was 2.1% Ce6 found on the UC surfae (upplementary Fig. 5). In priniple, ellular uptake of the peptide and Ce6-onjugated UCs may first redue the disulfide bond of proteted ysteine due to the redutive ellular environment. The subsequent lysosomal enzyme proessing will leave the peptide and expose a free 1,2-aminothiol group in ysteine, whih easily undergoes ondensation reation with C moiety in CBT struture 13,18, and thus triggers the loalization of ross-linked UCs at the tumour region (upplementary Fig. 6). During the reation, great are needs to be taken to avoid nonspeifi ross-linking by providing optimal spaing in the enzyme-responsive peptide and the short PEG linker to ontrol the distane between the peptide and partile surfae. First, we examined the ovalent ross-linking reations of upon enzyme treatment in buffer through partile morphology and spetrosopi analysis. After inubation of (1.5 mg ml 1 ) with CtsB (55 nm), transmission eletron mirosopy and dynami light sattering analysis indiated signifiant partile aggregation in solutions (Fig. 2a,b and upplementary Fig. 7), while alone or the enzyme-treated together with CtsB inhibitor (antipain), remained unhanged over time. Furthermore, the absorption spetrum showed a new peak at 489 nm during the enzyme reation, indiating the formation of firefly luiferin struture in solutions 13. These results learly suggest that the partiles 2 ATURE COMMUICATIO 7:10432 DOI: /nomms

3 ATURE COMMUICATIO DOI: /nomms10432 ARTICLE C F K C C (tbu) UC C K F (tbu) 808 nm light C C F K C (tbu) Chlorin e6 (Ce6) UC C(tBu) C K F Lysosome Enzyme-triggered loalization UCLEU Covalent partile ross-linking 1 O 2 Enhaned light onverting to amplify 1 O 2 prodution Figure 1 Illustration of the miroenvironment-sensitive strategy for ovalent ross-linking of peptide-premodified UCs in tumour areas. Upon tumour-speifi athepsin B (CtsB) enzyme reations, the peptide leavage on the partile surfae indues ovalent ross-linking between the exposed free ysteine and 2-yanobenzothiazole on neighbouring partiles, whih triggers the aumulation of UCs into the tumour site. uh enzyme-triggered ovalent ross-linking of UCs leads to an enhaned uponversion emission after 808 nm laser irradiation, and in turn amplifies the singlet oxygen generation from the photosensitizers (for example, Ce6) attahed on UCs for enhaned PDT treatment in vitro and in vivo. aggregation is mainly due to the speifi CtsB enzyme-triggered ross-linking reation (upplementary Fig. 8). Compared with without CtsB treatment, the ovalent ross-linking of ould lead to an enhaned uponversion luminesene. There was up to 2.2-fold luminesene emission intensity enhanement observed at 655 nm after 4 h reation (Fig. 2, d and upplementary Fig. 9). imilar enzyme-triggered ross-linking was also displayed by the luminesene olour hange in the absene and presene of CtsB inhibitor (upplementary Fig. 10), further onfirming that it is the speifi enzyme reation that indues the enhaned luminesene from partiles. Considering deep tissue penetration apaity and great advantages of the photoaousti imaging tehnique that integrates both funtions of ultrasound and optial imaging, we assessed the differene in photoaousti signal during the speifi enzyme ross-linking reations. Contrary to the inreased luminesene observed, there was an obvious photoaousti phantom signal derease (B40% in intensity) deteted in solution after 4 h reation (Fig. 2d). Generally, laser illuminations on enzymeresponsive UC onjugates would exite the internal energy levels of lanthanide dopants. The exited states an relax to the ground state through both radiative and nonradiative proesses. The enhaned radiative emission relaxation may orrespondingly lead to a deativation of the nonradiative relaxation in photoaousti, therefore resulting in the dereased photoaousti signals as observed in the experiments These results demonstrate that suh inverse trend observed in photoaousti may also be attributed to the speifi enzyme-triggered rosslinking reation. Furthermore, we also investigated the underlying reason of the enzyme-responsive luminesene enhanement of by monitoring the deays of the luminesene lifetime. As shown in Fig. 2e, the presene of Ce6 on partiles would lead to a sharp deay (from 500 to 205 ms) in lifetime at 655 nm, indiating the strong absorption of Ce6 at around 660 nm (ref. 35). Moreover, upon enzyme treatment, there was no signifiant lifetime hange of lanthanide dopants in (Fig. 2f and upplementary Fig. 11). In addition, we also examined the resonane light sattering properties of before and after CstB reations (upplementary Fig. 12). There was obvious sattering inrement observed upon 808 nm laser exitation, mostly due to the ovalent ross-linking of UC onjugates triggered by enzyme reation. The inrease in sattering effiieny enhaned the amount of light absorption among the ross-linked partiles, whih ould be the possible fator ontributing to the inreased uponversion luminesene Enzyme-triggered ross-linking of UC in living ells. The intraellular uptake and enzyme-triggered ross-linking of UCs in living ells were further investigated by fluoresene and photoaousti imaging tehniques. Typially, CtsB-overexpressing HT-29 tumour ell and CtsB-defiient IH/3T3 ells were hosen as models to inubate with enzyme-responsive (50 mgml 1 )at37 Cfor 4 h. The different expression levels of CtsB were verified by western blotting and quantitative human CtsB ELIA kit in HT-29 (40.1±4.4 ng mg 1 protein) and IH/3T3 (2.2±1.4 ng mg 1 protein) ells, respetively (upplementary Fig. 13) 44. As shown in Fig. 2g, the obvious yellow fluoresene ontributed from light-uponversion emissions at 545 and 655 nm was learly observed in the HT-29 ells, indiating the effetive ellular internalization of. Moreover, ompared with the ells inubated with CtsB inhibitor or ontrol onjugates (the ontrol partiles with surfae modified by non-ross-linking A-FKC(tBu)AC sequene), HT-29 ells treated with enzyme-responsive exhibited an inreased luminesene. In addition, there was no obvious fluoresene enhanement deteted in CtsB-defiient IH/3T3 ells after inubation (upplementary Figs 14,15). These results learly suggested that the overexpressed CtsB enzyme in HT-29 ell ould indue ovalent ross-linking of and suh rosslinking leads to the desired partile loalization in the tumour miroenvironment. imilar ellular studies based on photoaousti imaging were also arried out to monitor loalization in living ells. As shown in Fig. 2h, the enzyme-responsive, when inubated with HT-29 ells, exhibited less photoaousti signal (B56%) than that inubated with IH/3T3 ells (CtsB-defiient). Moreover, proessing of in HT-29 ells upon bloking the CtsB ativity with inhibitors prevented the derease in photoaousti signal, orresponding to the speifi enzyme reativity. Moreover, inubation of with IH/3T3 ells would not indue obvious photoaousti signal hange, further onfirming that the enzyme proessing an ause the loalization of partiles within HT-29 ells and the enhaned uponverting emissions resulted in the photoaousti signal derease. This was in agreement with the observation tested in buffer. We also evaluated the apability of to generate ytotoxi singlet oxygen ( 1 O 2 ) through the standard singlet oxygen sensor green 45. Basially, 808 nm laser irradiation of onjugates resulted in the onverted emission 655 nm, orresponding to the absorption of photosensitizer Ce6 labelled on UCs surfae. Compared with alone or enzyme-treated with inhibitor, the CtsB enzyme-indued ross-linking an ause ATURE COMMUICATIO 7:10432 DOI: /nomms

4 ATURE COMMUICATIO DOI: /nomms10432 a b d umber (%) ,200 1,800 ize (nm) ize (nm) Before ross-linking After ross-linking umber (%) PL intensity (a.u.) I 15/2 2 H 11/2 4 0 h I 4 15/2 I 1 h 15/2 2 h 4 3 h 4 3/2 F 4 h 9/ Wavelength (nm) PA signal (a.u.) PA signal Fluoresene signal CtsB PL intensity (a.u.) e f g DAPI Ce6 UC Merge Log (intensity) Fit 500 μs 205 μs PEI/PAA@UCs Ce6-PEI/PAA@UCs Time (ms) 2.0 Log (intensity) Fit 220 μs 245 μs CtsB ( ) CtsB (+) Time (ms) +inhibitor (ontrol) h HT-29 Inhibitor + PA signal IH/3T3 + i PL intensity (a.u.) CtsB ( ) CtsB (+) CtsB (+) with tissue 808 nm 660 nm j Cell viability (%) IR ( ) IR (+) IR (+) Conentration (μg ml 1 ) Figure 2 In vitro studies of tumour-speifi enzyme (CtsB) triggered ross-linking of upon 808 nm illuminations. TEM and DL analysis of ovalent ross-linking of (1.5 mg ml 1 ) without (a) and with (b) CtsB (55 nm) at 4 h. The hydrodynami diameters in DL are about 110 nm (a) and 1,580 nm (b). ale bar, 200 nm. () UCL of at different time intervals with CtsB treatment. The inset shows the luminesene photograph of inubate with CtsB for 4 h. (d) Photoaousti (PA) imaging and UCL signal after 4 h inubation of with CtsB. (e) Luminesene deay urves of the emission at 655 nm of UCs in the absene and presene of Ce6 exited by 808 nm laser. (f) Lifetime of in the absene and presene of CtsB treatment. (g) Confoal imaging of HT-29 ells inubated with, and inhibitor, and ontrol, respetively. Blue: DAPI (l ex ¼ 350/50 nm, l em ¼ 460/50 nm), red: Ce6 (l ex ¼ 545/25 nm, l em ¼ 610/75 nm), yellow: UC (l ex ¼ 980 nm, l em ¼ nm). ale bar, 20 mm. (h) PA signals of HT-29 and IH/3T3 living ells without (left) and with (right) CtsB inhibitor. The PA signals were shown with a laser exitation at 680 nm. (i) Quantifiation of singlet oxygen generation before and after enzyme-triggered or overed by 2-mm thik pork tissue when exposed to 808 or 660 nm laser irradiation for 1 h (0.4 Wm 2 ). (j) Cell viability of HT-29 at different onentrations of and after IR light treatment for 1 h. Data are mean±s.d. (n ¼ 3 tehnial repliates). enhaned light onverting emission, whih thus amplifies the prodution of 1 O 2 from the ativated photosensitizers after 1 h illumination (Fig. 2i and upplementary Fig. 16). Importantly, a 2-mm thik pork tissue (adipose tissue) was also used to mimi linial skin for further investigation of the different penetration of 808 nm (IR) and 660 nm light irradiation. As shown in Fig. 2i, the presene of pork tissue signifiantly dereased the effiieny of 1 O 2 generation (2.9-fold derease) with 660 nm irradiation, while there was only 0.6-fold derease in 1 O 2 generation when illuminated at 808 nm. The higher singlet oxygen prodution with 808 nm exitation in the presene of pork tissue learly indiated a better penetration depth as ompared with irradiation at 660 nm, whih makes UC-based PDT suitable for effetive tumour treatment in living system. The potential photodynami ytotoxiity of was further evaluated through an in vitro toxiology assay (TOX8). As shown in Fig. 2j, there was negligible ytotoxiity in the -inubated HT-29 ell without laser exposure at 808 nm, suggesting minimum toxiity aused by the partile itself. However, upon 808 nm laser illumination of (200 mgml 1 ) inubated HT-29 ells, there was higher ytotoxiity (B53% ell viability) observed in HT-29 ells than that of ells treated with the ontrol (B64% ell viability). In addition, similar photodynami inativation was also studied in -inubated HT-29 ells but with CtsB inhibitor or in the negative ontrol (CtsB-defiient IH/3T3 ells). Compared with the laser-exited HT-29 ells with, there was no signifiant enhanement of ytotoxiity deteted in these ontrol experiments. Meanwhile, 808 nm laser irradiation alone did not indue obvious ytotoxiity in both HT-29 and IH/3T3 ells (upplementary Fig. 17), indiating that the ovalent rosslinking of triggered by speifi enzyme would inrease the singlet oxygen generation, and thus indue the enhaned ell death in the targeted tumour ells with overexpression of the 4 ATURE COMMUICATIO 7:10432 DOI: /nomms

5 ATURE COMMUICATIO DOI: /nomms10432 ARTICLE CstB enzyme. Moreover, the ytotoxiity of HT-29 ells inubated with was also evaluated upon 660 or 808 nm light irradiation. Although the photodynami ytotoxiity was found to be higher at diret 660 nm exitation (B86% in ytotoxity) than that at 808 nm irradiation (B39% in ytotoxity) (upplementary Fig. 18), the presene of pork tissue an signifiantly lower the ytotoxiity at 660 nm illumination (B13%,B6.6-fold derease), while only less ytotoxiity dereases at 808 nm irradiation (B28%,B1.4-fold derease). uh ytotoxiity studies were onsistent with the trends of 1 O 2 generation as above, learly demonstrating the great advantage of deep-tissue penetration of 808 nm (IR) light for living ell PDT treatment. In vivo ross-linking of in tumour-bearing mie. Inspired by the promising imaging and PDT results in living ells, we examined the in vivo theranosti effiay via intratumoural injetion of into living female Balb/ nude mie. We implanted HT-29 ells (CtsB overexpressing) into the left and right flanks of nude mie. At 2 3 weeks post-implantation, the tumours grew to 3 5 mm in diameter and the living mie underwent photoaousti imaging and PDT studies. Typially, one group of xenograft mie bearing two tumours were subjeted to and ontrolled onjugates, respetively (3 mg in 100 ml saline), followed by photoaousti imaging. As ompared with the photoaousti intensity before injetion, the mie exhibited a signifiant inrease in photoaousti signals at 4 h post-injetions for both onjugates. Moreover, the photoaousti signals in the tumour injeted with were found lower (B30% derease in intensity) than that subjeted to (Fig. 3a and upplementary Fig. 19). These results suggested the feasibility of the enzyme proessing to trigger the loalization of at the tumour site and it was the ovalent ross-linking that led to the dereased photoaousti signals. Furthermore, we also investigated the therapeuti effet of as a PDT agent for tumour therapy in living mie. As shown in Fig. 3b, in group 1 (n ¼ 8), was injeted into the implanted tumour on the left flank, and followed by an 808 nm laser irradiation at the tumours after 4 h injetion. As ontrast, group 2 (n ¼ 8) mie were intratumourally injeted with the ontrol on the right flank and subsequent light exposure under idential onditions. Tumour-bearing mie with diret injetion of but no IR light irradiation were assigned as group 3 (n ¼ 8). The last group (group 4, n ¼ 8) onsisted of mie injeted with saline only. The tumour sizes in the different groups were measured to assess the PDT effiay over a period of 2 weeks. After -mediated PDT treatment (P ¼ 0.038), the tumour growth in group 1 had been signifiantly inhibited as ompared with that observed in group 2 with ontrol (P ¼ 0.033, Fig. 3), indiating that the enzyme-triggered ross-linking of UC partiles at the tumour region had an important role in enhaning therapeuti outomes. On the other hand, in group 3 (P ¼ 0.021) and 4, the tumour sizes grew exponentially over the period of time, suggesting that the treatment with 808 nm laser only or without light irradiation indued minimum inhibition of tumour growth. To further evaluate the therapeuti effiay, we also performed TUEL staining on the tissue setions from different groups after 11 days of PDT treatment (Fig. 3d and upplementary Fig. 20). As expeted, the treated tumours in group 1 mie displayed signifiant damage than those in group 2 mie. The mie treated with IR light alone (group 4) and probe without light (group 3) displayed no detetable damage. This is in good aordane with the trends observed in tumour inhibition after IR light-mediated PDT treatment. More importantly, there were no signifiant burnt sars on the tumour skins in similar light-mediated treatment by onventionally used 980 nm laser illuminations, learly implying that the novel enzyme-responsive onjugates with laser irradiation at 808 nm ould serve as a reliable platform for effetive in vivo antitumour treatment. In vivo PDT treatment with affinity ligand-modified. To further improve the tumour uptake and PDT effiieny, we onjugate an affinity ligand, foli aid (FA), to the surfae of a b PA signal (a.u.) 9,000 6,000 3,000 0 h 4 h Time after injetion (h) Group 1 Group 2 Group 3 Group 4 Relative tumour volume (V/V 0 ) Group 1: +IR Group 2: +IR Group 3: Group 4: aline+ir * * * Time after treatment (day) d IR (+) IR (+) IR ( ) aline IR (+) Figure 3 In vivo photoaousti imaging and PDT effiieny by intratumoural injetion of. (a) PA signals in tumour region after injetion of (3 mg in 100 ml saline). (b) Photographs of tumour-bearing mie after 11 days treatment with (group 1), (group 2), without IR light irradiation (group 3) and saline (group 4). () Relative tumour volumes in the treated groups after PDT therapy under 808 nm laser irradiation. The treatment after 4 h of intratumoural injetion (3 mg in 100 ml saline) was started on day 0 and repeated twie at day 4 and day 8 (arrows) followed by 808 nm laser irradiation. (d) TUEL histology of tumour tissues after IR light-mediated tumour treatment. ale bar, 100 mm. Date are means±s.d. (n ¼ 8 mie per group). tatistial signifiane is assessed by a tudent s t-test (heterosedasti, two-sided). *Po0.05. ATURE COMMUICATIO 7:10432 DOI: /nomms

6 ATURE COMMUICATIO DOI: /nomms10432 for their reognition to folate reeptors that overexpress in most tumours. Meanwhile, onsidering the fat to enhane the bioompatibility of in living system, the FA was first oupled with polyethene glyol (PEG 5000 ) spaer ontaining amino and thiol group at eah terminus. uh FA-PEG H linkers were then oupled to enzyme-responsive onjugate through H-dPEG 2 -Mal as a linkage moiety to afford final produt of FA-PEG@ (Fig. 4a and upplementary Fig. 21). Upon reation with CtsB enzyme, FA-PEG@ indiated similar luminesene enhanement as ompared with previous platform without FA modifiation, suggesting that introdution of FA and PEG linker into platform would not influene its ativity for enzyme reognition (upplementary Fig. 22). To study whether FA onjugation an aid tumour uptake in living mie, we used fluoresene imaging to real-time monitor the targeting effet in vivo. The female Balb/ nude mie bearing HT-29 tumours were divided into three groups. Group 1 onsisted of the mie with intravenous injetion of saline. The mie subjeted to PEG-modified partiles but no FA ligands were assigned to group 2. In group 3, the tumour-bearing mie were injeted with FA-PEG@ via the tail vein. The fluoresent images were reorded at different time intervals after the first injetion. Relative to ontrol studies, a signifiant inrease in fluoresene was observed in living mie at 4 h post-injetion of probes (Fig. 4b and upplementary Fig. 23). In ontrast, the tumours in group 3 with injetion of FA-PEG@ displayed stronger fluoresene than that in group 2 subjeted to PEG@ injetion. Both fluoresene signals gradually dereased 24 h post-administration through the proess of irulation. In addition, similar tumour targeting was also validated by photoaousti imaging in living mie. After administration of onjugates at 1 h, the mie showed obvious photoaousti signal inrease in tumours. Moreover, the observed photoaousti signals in the tumours of group 3 were higher than that in group 2 (Fig. 4). The higher fluoresene and photoaousti signals present in tumours onfirmed the possible effiay of the FA ligand in improving the targeting of onjugates at the tumour region. Enouraged by the imaging data that revealed the effetive tumour uptake of partiles, we ontinued the lightmediated PDT therapy via intravenous injetion of surfaemodified onjugates or saline into six different groups of mie, and in vivo tumour inhibition effet was evaluated upon 808 or 660 nm light illumination. As shown in Fig. 4d, after 808 nm laser illumination, the tumour sizes in group 2 (n ¼ 8, P ¼ 0.050, with injetion of PEG@) and 3 (n ¼ 8, P ¼ 0.038, injeted with FA-PEG@) were found to be redued signifiantly over the therapeuti period, whereas the tumour sizes in ontrol group 1 (n ¼ 8) with injetion of saline revealed a fast growth rate. In addition, during the treatment, the tumours in group 3 displayed more effetive inhibition than those in group 2. These data suggested that the failitated tumour uptake by FA ligands and effetive tumour redution was mainly a b 0 h 4 h 8 h 24 h C F K (tbu) (tbu) C K F C FA-PEG urfae labeling F K (tbu) PEG-FA FA-PEG@ K F (tbu) C PEG-FA PEG@ FA-PEG@ e 0 h 1 h 0 h 1 h PA signal PA signal h 1 h 0 h 1 h d Relative tumour volume (V/V 0 ) aline PEG@ 808 nm FA-PEG@ 808 nm FA-PEG@ 660 nm FA-PEG@ 808 nm+tissue FA-PEG@ 660 nm+tissue Time after treatment (day) ** * * * Tumour targeting in living mie H&E staining aline IR (+) PEG@ IR (+) FA-PEG@ IR (+) Figure 4 In vivo targeted tumour imaging and PDT therapy by intravenous injetion of FA-PEG@. (a) Illustration of the targeting strategy of FA-PEG@ through the onjugation of FA and PEG 5000 on. (b) Fluoresene imaging of tumours (blue irle) in living mie at different time intervals after injetion (from left to right: aline, PEG@, FA-PEG@, respetively; l ex ¼ 640 nm, l em ¼ 670 nm). () PA imaging signals in the tumour region at different time intervals after intravenous injetion (top: PEG@, bottom: FA-PEG@, 3 mg in 100 ml saline). (d) Tumour volumes hange as a funtion of time in treated groups to evaluate the effetiveness of PDT treatment in vivo. The PDT treatment was arried out after 4 h drug-light interval of intravenous injetion (3 mg in 100 ml saline) and periodially repeated at indiated time points (arrows) following by 808 nm laser irradiation every two days. (e) H&E histology of tumour tissues after 808 nm mediated tumour treatment by intravenous injetion. ale bar, 200 mm. Date are means±s.d. (n ¼ 8 mie per group). tatistial signifiane is assessed by a tudent s t-test (heterosedasti, two-sided). *Po0.05, **Po ATURE COMMUICATIO 7:10432 DOI: /nomms

7 ATURE COMMUICATIO DOI: /nomms10432 ARTICLE from the prodution of 1 O 2 from Ce6 anhored on the surfae. otably, diret 808 nm (group 3) and 660 nm (group 5, P ¼ 0.008) laser irradiation of living animals with intravenous injetion of FA-PEG@ resulted in omparable PDT effiieny, in whih tumour growth has been greatly suppressed (Fig. 4d). However, onsidering the different light penetration, similar in vivo PDT treatments were also performed when the tumours were overed with a 2-mm thik pork tissue. Compared with mie with FA-PEG@ injetion and laser illumination at 808 nm (group 4, P ¼ 0.019), the presene of pork tissue ould signifiantly ompromise tumour therapy for the mie treated with FA-PEG@ and 660 nm laser exitation (group 6, P ¼ 0.029), further demonstrating the benefits of deep tissue penetration in 808 nm laser-mediated tumour treatment. Besides the promising antitumour therapy in vivo, the histologial tumour tissues analysis also revealed the effiay of PDT treatment. After 12 days of light-mediated PDT at 808 nm, hematoxylin and eosin and TUEL staining of tumour tissues displayed more signifiant damage in the mie with intravenous injetion of FA-PEG@ (group 3) than those tumours treatment in ontrol groups (Fig. 4e and upplementary Fig. 24). Moreover, there was no obvious damage observed in other organs during the treatment (upplementary Fig. 25). And our indutively oupled plasma spetral analysis also validated more Y 3 þ ion distribution in the tumour region (upplementary Fig. 26). These results learly indiated that onjugates modified with ative targeting agents an signifiantly improve the therapeuti effiay of in vivo tumour treatment with a desirable PDT outome. Disussion Currently, nanomediine based on lanthanide-doped UCs towards aner imaging and therapy has reeived onsiderable attention due to their intrinsi photon-onverting nature upon exposure to long-wavelength light irradiation Despite suh exeptional optial apabilities in the IR ranges offered by UC nanopartiles, the ability to provide speifi targeting strategy for effetive loalization of UCs within pathologial areas, espeially at the tumour site, remains a ritial hallenge. o far, the ligand-mediated reognition strategies based on the onjugation of affinity moieties to the partile surfae have been mostly applied to ahieve tumour targeting speifiity Considering the omplex and dynami physiologial ell onditions, the results along with the stohasti nature of ligand reeptor interations between normal and tumour ells are not so satisfatory. Alternative to develop smart onjugating nanoplatforms that are sensitive to loal tumour environment onditions (for example, a partiular ph, temperature or enzyme level and so on), and then failitate speifi loalization of UC partiles at tumour sites would be thus a highly desirable option 10,11. In this study, by exploiting the advantages of intrinsi enzymati stimulus to speifially target the tumour areas, we developed suh a smart uponversion nanoonjugate that an seletively reat with protease enzyme CtsB, whih has been onsidered as one important biomarker in many types and stages of aners 37,44. Upon tumour-speifi enzymati CtsB reation, the CtsB-sensitive peptide on the UC partile surfae an be leaved, whih exposes free ysteine and indues its ondensation reation with CBT groups on the surfae of neighbouring partiles, therefore triggering the ovalent ross-linking of UCs partiles. Our extensive in vitro and in vivo fluoresene and photoaousti imaging studies have validated the suessful enzyme-triggered ross-linking reation, whih easily reognized the enhaned light onverting emission and unique targeting of enzyme-sensitive UCs at tumour sites. o far, photodynami antitumour therapy has been regarded as a minimally invasive treatment modality, and has been widely applied in linis for various types of aner treatment 28,30,32. Compared with onventional hemotherapy, light-mediated PDT exhibits some unique advantages, in whih ytotoxi photosensitizers an be seletively ativated by manipulating the loation of light exposure. Currently, the most linially used photosensitizers are exited at a shorter wavelength range from 630 to 700 nm, whih unfortunately has limited penetration depth in biologial tissues. One promising strategy to avoid this limitation is to shift the exitation wavelength to the IR region (700 1,000 nm), in whih biologial tissues have maximum light transpareny, and thus ideal for in vivo optial imaging and phototherapy 35,36. Atually, latest advanes in rareearth doped UCs have witnessed unique uponverting optial properties, whih an transdue low-energy IR light illumination into high-energy emissions from ultraviolet to visible range. uh exeptional light-onverting apabilities ould greatly math the demands and thus ahieve deeper tissue penetration to benefit biomedial appliations inluding photodynami therapy, biologial imaging and remotely ontrolled release of therapeuti agents in vitro and in vivo 5,27 30,32. Current progress of UC-based PDT system mostly utilizes the IR light exitation wavelength at 980 nm, at whih, however, the absorption of water is signifiant uh 980 nm lightmediated PDT is faing tremendous hurdles to travel into deeper tissue and bypass the inevitable heat effet Therefore, efforts have been intensively made to adjust the exitation range of UCs to a shorter wavelength to meet the demands of the medial spetral window (that is, B nm) (refs 35,36). Very reently, d 3 þ -doped uponversion proesses based on a new exitation wavelength around 808 nm have been investigated to manipulate optial properties of lanthanide UCs to minimize this photothermal effet 26,36. By taking this promising advantage, in this study, we have arried out a series of UC-based PDT experiments, and systematially ompared the differenes of photodynami antitumour effet in vitro and in vivo through 808 and 660 nm laser irradiation. Our data learly demonstrated that the PDT treatment effiay upon 808 nm light illumination did not show signifiant derease when the pork tissue (for example, 2 mm thikness) was used to inrease the tissue depth; however, the dramati drop of therapeuti effet was observed in the similar treatment under 660 nm laser exitation (Figs 2i and 4d, and upplementary Fig. 18), suggesting that 808 nm lightmediated UCs exhibit deeper tissue penetration and better antitumour effet than those in the onventional PDT treatment system. Meanwhile, during the proess of effetive UC-based PDT treatment, the prolonged 808 nm laser irradiation would not indue signifiant overheating and tissue damage, thus providing promising prospet for their safer appliations in the future. More signifiantly, in response to speifi enzyme stimulation within tumours, our unique UCs platform ould undergo ovalent ross-linking reations and enable the seletive aumulation of partiles at the tumour region. The in situ enzyme-triggered interpartile ross-linking exhibited enhaned light onverting emission and ould amplify the singlet oxygen generation. Both our in vitro and in vivo theranosti studies suessfully demonstrated that suh unique enzyme-responsive loalization of light-onverted rare-earth nanostrutures ould signifiantly inhibit the tumour growth in proess of PDT treatment as ompared with those UCs that ould not undergo ross-linking reation. In summary, a novel targeting strategy based on speifi enzymati reation was presented to effetively loalize light ATURE COMMUICATIO 7:10432 DOI: /nomms

8 ATURE COMMUICATIO DOI: /nomms10432 onverting UC nanoarriers at tumour area. uh tumour miroenvironment-responsive enzyme reations would trigger the ovalent ross-linking of single UC partiles, and thus display unique partile loalization to failitate promising multimodality imaging and amplified PDT therapy of malignant tumours in vitro and in vivo upon IR light illumination at 808 nm. This well-defined rational design ould be easily extended into other unique funtional materials and open new doors for preision nanomediine in the future linial appliations. Methods General. yntheti proedures and hemial haraterizations of all the probes are desribed in the upplementary Methods. In vitro fluoresene imaging in living ells. The human oloretal adenoarinoma ell line (HT-29, at. no. HTB-38) and mouse embryoni fibroblast ell line (IH/3T3, at. no. CRL-1658) were provided from Amerian type ulture olletion and heked for myoplasma ontamination. These ells are not listed by ICLAC as misidentified ell lines (3 Otober 2014). CtsB-overexpressing HT-29 ells and CtsB-defiient IH/3T3 ells were seeded with a ell density of in an ibidi onfoal m-dish (35 mm, plasti bottom) in 1 ml DMEM media whih was inubated for 24 h at 37 C. The seeded ells were subsequently treated with (50 mgml 1 ) and inubated for 4 h. The ell nulei were stained by DAPI (1 mgml 1 ) for 20 min and then the ells were washed with PB (ph 7.4) for three times. Control experiments were setup by treating the two ell lines with in the presene of CtsB inhibitor and ontrolled onjugates. For the ell-inhibition assay, the ells were first pretreated with CtsB inhibitor (antipain hydrohloride, 100 mm in DMEM) for 2 h at 37 C followed by the addition of and inubated for further 4 h. Fluoresene imaging of the samples were then arried out under onfoal mirosopy system using a ontinuous-wave 980 nm laser as exitation soure (EIT Tehnology). The quantifiation of CtsB enzyme expression in HT-29 and IH/3T3 ells were performed by Human CtsB ELIA kit aording to the standard proedure desribed by the manufaturer (CUABIO, UA). In vivo animal studies. All animal experimental proedures were performed in aordane with the protool # approved by the Institutional Animal Care and Use Committee (IACUC). Female Balb/ nude mie (B6 8-weeks old) were purhased from Charles River Laboratories (hanghai, China). Xenograft mie models were established by injeting 0.1 ml of tumour ells suspension (PB/matrigel, BD biosienes, 1:1 v/v) ontaining HT-29 ells into both flanks or only right side of mouse. When the tumour volume reahed a palpable size, the mouse was used for further studies. In vivo ross-linking of in tumour-bearing mie. Xenograft mie models were treated when the tumour volumes approahed 3 5 mm in diameter. For in vivo PDT treatment, the tumour-bearing mie were randomized into four groups (n ¼ 8, eah group) and treated by intratumoural injetion of partile samples. In group 1, (3 mg in 100 ml of saline) was diretly injeted into implanted tumours, followed by an 808 nm laser irradiation after 4 h injetion. In group 2, same amounts of ontrolled onjugates were injeted with subsequent laser exposure. Control experiments with injetion of but no IR light irradiation (group 3) and saline with laser treatment (group 4) were used, respetively. The effetive exposure time for eah mouse was 45 min (5 min interval after 5 min irradiation with total treatment time of 90 min) with a power density at 0.4 W m 2 (fluene 1,080 J m 2 ). A seond- and third-dose injetion followed by PDT treatment desribed above was repeated at 4 days and 8 days after the first-dose injetion, respetively. Tumour size was measured three times a week using a vernier aliper upon PDT treatment. The tumour volume was alulated using the following equation: tumour volume (V) ¼ length width 2 /2. Relative tumour volume was alulated as V/V 0 (V 0 was the initial tumour volume before PDT treatment). In vivo targeted PDT treatment with intravenous injetion. Xenograft HT-29 tumour model on the right side of female Balb/ mie were first developed as desribed above. For in vivo tumour targeting PDT treatment, the tumour-bearing mie had been randomly arranged into six different groups (n ¼ 8, eah group) to perform a series of intravenous injetions of saline (group 1), PEG@ (group 2) and FA-PEG@ (group 3 6), respetively (3 mg in 100 ml saline). After 4 h of intravenous injetion, laser treatment was performed on groups 1 6 by irradiating the tumour region with a ontinuous 808 or 660 nm laser. The effetive exposure time for eah mouse was 45 min (5 min interval after 5 min irradiation with a total treatment time of 90 min) with a power density at 0.4 W m 2 (fluene 1,080 J m 2 ). The tumours in group 4 and 6 were overed by 2-mm thik pork tissue and then irradiated with 808 or 660 nm lasers under the same ondition. The PDT treatment with laser irradiation was repeated every 2 days. A seond- and third-dose injetion of the PDT treatment desribed above was repeated at 4 days and 8 days after the first-dose injetion, respetively. Tumour size was measured and alulated as desribed above. Referenes 1. Gao, Y., hi, J., Yuan, D. & Xu, B. Imaging enzyme-triggered self-assembly of small moleules inside live ells. at. Commun. 3, (2012). 2. Williams, R. J. et al. Enzyme assisted self-assembly under thermodynami ontrol. at. anotehnol. 4, (2009). 3. Lovell, J. F. et al. Porphysomenanoesiles generated by porphyrin bilayers for use as multimodal biophotoni ontrast agents. at. Mater. 10, (2011). 4. Petros, R. A. & Desimone, M. J. trategies in the design of nanopartiles for therapeuti appliations. at. Rev. Drug Disov. 9, (2010). 5. Mitragotri,., Burke, P. A. & Langer, R. Overoming the hallenges in administrating biopharmaeutials: formulation and delivery strategies. at. Rev. Drug Disov. 13, (2014). 6. Cheng, C. J., Tietjen, G. T., auier-sawyer, J. K. & altzman, W. M. A holisti approah to targeting disease with polymeri nanopartiles. at. Rev. Drug Disov. 14, (2015). 7. Davis, M. E., Chen, Z. G. & hin, D. M. anopartile therapeutis: an emerging treatment modality for aner. at. Rev. Drug Disov. 7, (2008). 8. Giljohann, D. A. & Mirkin, C. A. Drivers of biodiagnostis development. ature 580, (2009). 9. Koo, H. et al. In vivo targeted delivery of nanopartiles for theranosis. A. Chem. Res. 44, (2011). 10. Yu, M. K., Park, J. & Jon,. Y. Targeting strategies for multifuntional nanopartiles in aner imaging and therapy. Theranostis 2, 3 44 (2012). 11. Thaker, A.. & Gambhir,.. anoonology: the future of aner diagnosis and therapy. Caner J. Clin. 63, (2013). 12. Mura,., iolas, J. & Couvreur, P. timuli-responsive nanoarriers for drug delivery. at. Mater. 12, (2013). 13. Liang, G., Ren, H. & Rao, J. A bioompatible ondensation reation for ontrolled assembly of nanostrutures in living ells. at. Chem. 2, (2010). 14. Haun, J. B., Devaraj,. K., Hilderbrand,. A., Lee, H. & Weissleder, R. Bioorthogonal Chemistry amplifies nanopartiles binding and enhanes the sensitivity of ell detetion. at. anotehnol. 5, (2010). 15. Tsukiji,., Miyagawa, M., Takaoka, Y., Tamura, T. & Hamahi, I. Ligand-direted tosyl hemistry for protein labeling in vivo. at. Chem. Biol. 5, (2009). 16. Laughlin,. T., Baskin, J. M., Amaher,. L. & Bertozzi, C. R. In vivo imaging of membrane-assoiated glyans in developing zebrafish. iene 320, (2008). 17. Presher, J. A., Dube, D. H. & Bertozzi, C. R. Chemial remodelling of ell surfaes in living animals. ature 430, (2004). 18. Ye, D. et al. Bioorthongonal ylization-mediated in situ self-assembly of small-moleule probes for imaging aspase ativity in vivo. at. Chem. 6, (2014). 19. Lang, K. & Chin, J. W. Cellular inorporation of unnatural amino aids and bioorthogonal labeling of proteins. Chem. Rev. 114, (2014). 20. Grammel, M. & Hang, H. C. Chemial reporters for biologial disovery. at. Chem. Biol. 9, (2013). 21. Deng, R. et al. Temporal full-olour tuning through non-steady-state uponversion. at. anotehnol. 10, (2015). 22. Feng, W., Zhu, X. & Li, F. Reent advanes in the optimization and funtionalization of uponversion nanomaterials for in vivo bioappliations. PG Asia Mater. 5, e75 (2013). 23. Yang, Y. et al. In vitro and in vivo unaging and bioluminesent imaging through photoaged uponversion nanopartiles. Angew. Chem. Intl. Ed. 51, (2012). 24. Idris,. M. et al. In vivo photodynami therapy using uponversion nanopartiles as remote-ontrolled nanotransduers. at. Med. 18, (2012). 25. Yang, D. et al. Current advanes in lanthanide ion (Ln 3 þ )-based uponversion nanomaterials for drug delivery. Chem. o. Rev. 44, (2015). 26. Xie, X. J. et al. Mehanisti investigation of photon uponversion in d 3 þ -sensitized ore-shell nanopartiles. J. Am. Chem. o. 135, (2013). 27. Kahynski, A. V. et al. Photodynami therapy by in situ nonlinear photon onversion. at. Photon. 8, (2014). 28. Xu, C. T. et al. Uponverting nanopartiles for pre-linial diffuse optial imaging, mirosopy and sensing: urrent trends and future hallenges. Laser Photon. Rev. 7, (2013). 29. Zou, W., Visser, C., Maduro, J. A., Pshenihnikov, M.. & Hummelen, J. C. Broadband dye-sensitized uponversion of near-infrared light. at. Photon. 6, (2012). 30. Wang, C., Cheng, L. & Liu, Z. Drug delivery with uponversion nanopartiles for multi-funtional targeted aner ell imaging and therapy. Biomaterials 32, (2011). 8 ATURE COMMUICATIO 7:10432 DOI: /nomms

9 ATURE COMMUICATIO DOI: /nomms10432 ARTICLE 31. Zhou, L. et al. ingle-band uponversion nanoprobes for multiplexed simultaneous in situ moleular mapping of aner biomarkers. at. Commun. 6, 6938 (2015). 32. Lu,. et al. Multifuntional nano-bioprobes based on rattle-strutured uponverting luminesent nanopartiles. Angew. Chem. Int. Ed. 54, (2015). 33. Xiao, Q. et al. A ore/satellite multifuntional nanotheranosti for in vivo imaging and tumor eradiation by radiation/photothermal synergisti therapy. J. Am. Chem. o. 135, (2013). 34. Li, L. et al. Biomimeti surfae engineering of lanthanide-doped uponversion nanopartiles as versatile bioprobes. Angew. Chem. Int. Ed. 51, (2012). 35. Ai, F. et al. A ore-shell-shell nanoplatform uponverting near-infrared light at 808 nm for luminesene imaging and photodynami therapy of aner. i. Rep. 5, (2015). 36. hen, J. et al. Engineering the uponversion nanopartile exitation wavelength: asade sensitization of tri-doped uponversion olloidal nanopartiles at 800 nm. Adv. Opt. Mater. 1, (2013). 37. Leaille, F., Kaleta, J. & Bromme, D. Human and parasiti papain-like ysteine proteases: their role in physiology and pathology and reent developments in inhibitor design. Chem. Rev. 102, (2002). 38. Wang, L. V. & Hu,. Photoaousti tomography: in vivo imaging from organelles to organs. iene 335, (2012). 39. ie, L. & Chen, X. trutural and funtional photoaousti moleular tomography aided by emerging ontrast agents. Chem. o. Rev. 43, (2014). 40. heng, Y., Liao, L. D., Thakor,. & Tan, M. C. Rare-earth doped partiles as dual-modality ontrast agent for minimally-invasive luminesene and dual-wavelength photoaousti imaging. i. Rep. 4, 6562 (2014). 41. Chris, H. J. H. et al. Multifuntional photosensitizer-based ontrast agents for photoaousti imaging. i. Rep. 4, 5342 (2014). 42. Atwater, H. A. & Polman, A. Plasmonis for improved photovoltai devies. at. Mater. 9, (2010). 43. Zeng, Z., Mizukami,., Fujitab, K. & Kikuhi, K. An enzyme-responsive metal-enhaned nearinfrared fluoresene sensor based on funtionalized gold nanopartiles. Chem. i. 6, (2015). 44. Talieria, M. et al. Cathepsin B and athepsin D expression in the progression of oloretal adenoma to arinoma. Caner Lett. 205, (2004). 45. Lovell, J. F., Liu, T. W. B., Chen, J. & Zheng, G. Ativatable photosensitizers for imaging and therapy. Chem. Rev. 110, (2010). Aknowledgements We thank Professor Lei Lu and Dr Meng hi for the Western blot assay. We also thank Xiangyang Wu, Fang Liu, Yang Zhang and Frane Widjaja for the helpful suggestions and disussions provided in data analysis. We aknowledge the finanial support from anyang Tehnologial University, ingapore, through a tart-up Grant (UG), Tier 1 RG (11/13), RG (64/10) and RG (35/15). The Major tate Basi Researh Development Program of China (973 Program) (Grant os. 2014CB and 2013CB733802), the ational atural iene Foundation of China (FC) (Grant os , and ), the Fundamental Researh Funds for the Central Universities, China (Grant o ), and the Program for ew Century Exellent Talents in University (CET ). Author ontributions X.A., J.A., J.M., and B.X. oneived the projets and are responsible for the design, experiments and preparation of the manusript. E.K.L.Y. assisted with spetrosopi measurement. X.A., J.A., C.J.H.H., A.B.E.A. and M.O. performed PA imaging and intratumoural theranosti study. Y.W. and X.L. ontributed to lifetime and quantum yield test. X.A., X.W., Y.W., H.C., M.G., X.C. and G.L. arried out in vivo imaging and intravenous PDT treatment analysis. All authors reviewed the manusript. Additional information upplementary Information aompanies this paper at natureommuniations Competing finanial interests: The authors delare no ompeting finanial interests. Reprints and permission information is available online at reprintsandpermissions/ How to ite this artile: Ai, X. et al. In vivo ovalent ross-linking of photon-onverted rare-earth nanostrutures for tumour loalization and theranostis. at. Commun. 7:10432 doi: /nomms10432 (2016). This work is liensed under a Creative Commons Attribution 4.0 International Liense. The images or other third party material in this artile are inluded in the artile s Creative Commons liense, unless indiated otherwise in the redit line; if the material is not inluded under the Creative Commons liense, users will need to obtain permission from the liense holder to reprodue the material. To view a opy of this liense, visit ATURE COMMUICATIO 7:10432 DOI: /nomms