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Theranostic colloidal nanoparticles of pyrrolopyrrole cyanine derivatives for simultaneous near-infrared fluorescence cancer imaging and photothermal therapy Mingfeng Wang, Shuo Huang, Wei Liu, Jing Huang, Xiaochen Wang, Cangjie Yang, Hassan Bohra, and Quan Liu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00321 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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ACS Applied Bio Materials

Theranostic colloidal nanoparticles of pyrrolopyrrole cyanine derivatives for simultaneous near-infrared fluorescence cancer imaging and photothermal therapy Shuo Huang, Wei Liu, Jing Huang, Xiaochen Wang, Cangjie Yang, Hassan Bohra, Quan Liu, * Mingfeng Wang* School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.

KEYWORDS: theranostic, cyanine, fluorescence, cancer, imaging, therapy

ABSTRACT Theranostic agents incorporating diagnostic and therapeutic agents together play crucial roles in clinical cancer treatment. On the one hand, near infrared (NIR) fluorescence imaging strategies are expected to provide high-resolution real-time structural and molecular information with deep penetration to biological tissues in vivo. On the other hand, photothermal therapy (PTT) offers highly efficient treatment to cancer with negligible safety concerns. To

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combine the strengths of both NIR fluorescence imaging and PTT for simultaneous cancer imaging and therapy, we report a type of NIR fluorescent nanoparticles (NPs) composed of pyrrolopyrrole cyanines (PPCys) with strong absorbance in the NIR optical window. These NPs as effective theranostic agents show strong NIR fluorescence and photothermal effect for simultaneous cancer imaging and therapy at both in vitro and in vivo levels. The in vivo imaging and therapy results in nude mice demonstrate the promising potential of these NIR NPs for preclinical and clinical cancer imaging and therapy applications.


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1. Introduction Molecules and nanomaterials with light absorption and emission in the NIR optical window (700-2500 nm) have attracted much attention for biomedical applications, particularly for tumor detection, image-guided surgery and phototherapy.1-10 The application of NIR light minimizes the interference caused by light scattering and autofluorescence from biological tissues, which benefits to achieve strong optical contrast in deep-tissue tumor imaging and high specificity in phototherapy with minimal side effects against normal cells and tissues.11-12 On the other hand, the achievement of these goals extremely relies on the selective targeting of NIR contrast agents to tumor tissues and cells. To date the phenomenon termed the enhanced permeability and retention (EPR) effect has been widely considered in design and synthesis of tumor-targeting imaging probes and therapeutic agents, despite some rebate of less convincing clinical evidences due to the extreme heterogeneity in human cancers compared to animal-based tumor models.13-14 Many solid tumors exhibit unique features such as extensive angiogenesis, defective vascular architecture

and

impaired

lymphatic

drainage/recovery

system.

As

a

consequence,

macromolecules or nanoparticles with nanoscale sizes (e.g. 30-200 nm) tend to selectively accumulate in tumors during blood circulation.15 Among a variety of NIR chromophores that have been reported, only indocyanine green (ICG) and methylene blue (MB) have been approved for clinical imaging applications.7 While both ICG and MB are hydrophilic and ionic which may induce some nonspecific interactions with biological species such as proteins, colloidal nanoparticles (NPs) of some hydrophobic NIR chromophores which are encapsulated by hydrophobic,16-17 hydrophilic18 or amphiphilic polymers19-25 have been reported. These NPs are either fluorescent or non-fluorescent, and have been used as the exogenous contrast agents for fluorescence-based optical imaging or light-


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absorption-based photoacoustic imaging and photothermal therapy. But photostable NIR contrast agents with both sufficient fluorescence intensity and photothermal effect, which is important for imaging-guided tumor surgery and/or treatment, still remain rare for simultaneous fluorescencebased imaging and photothemal therapy. Herein, we report a type of theranostic colloidal NPs composed of pyrrolopyrrole cyanine chromophores (denoted as PPCy-TPA) exhibiting both fluorescence for bioimaging in vivo and ex vivo and photothermal effect for the effective treatment of tumors in xenograft animal models. (Scheme 1) Co-precipitation of PPCy-TPA with Pluronic F127 as the stabilizer resulted in colloidal NPs with excellent stability and biocompatibility. In particular, PPCy-TPA NPs dispersed in water exhibited not only NIR-fluorescence but also high photothermal conversion (40%) under continuous laser (808 nm) irradiation. The in vivo penetration depth of these NIR NPs as contrast agents was examined in detail by fluorescence imaging of skin phantoms. The applications of PPCy-TPA NPs for NIR bioimaging and photothermal therapy were further demonstrated at both in vitro and in vivo levels. Moreover, the biodistribution of PPCy-TPA NPs was studied in a tumor model of nude mice, which was characterized non-invasively according to the detected NIR fluorescence intensity after intravenous injection of the nanoparticles. The in vivo tumor mice imaging results indicated that these PPCy-TPA NPs accumulated preferably in a tumor site presumably by passive targeting through a mechanism of enhanced permeability and retention (EPR) effect. The tumor-specific accumulation of the NPs enabled real-time monitoring of tumor progression by fluorescence imaging. Meanwhile, the tumor-bearing mice were effectively treated with the photothermal therapy using the PPCy-TPA NPs under 808-nm laser irradiation.


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Scheme 1. (a) Schematic illustration of chemical structure of PPCy-TPA derivative and preparation of PPCy-TPA NPs using an amphiphilic poly(ethylene glycol)100-b-poly(propylene glycol)65-b-poly(ethylene glycol)100 triblock copolymer (Pluronic 127) as the stabilizer, in which the blue block represents poly(ethylene glycol) (PEG) and the yellow block represents poly(propylene glycol) (PPG). (b) The application of PPCy-TPA NPs in NIR fluorescence imaging and photothermal therapy. 2. Materials and Methods Evaluation of photothermal effect, photostability, In-vitro photothermal study and In-vitro cytotoxicity were carried out with the same manner as in the previous report.21 2.1 Materials


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All chemicals were purchased from Sigma-Aldrich and used without further purification. Nude mice (C.Cg/AnNtac-Foxn1nunE9, male, 20 g in weight, aged 5-6 weeks) were obtained from InVivos Pte Ltd (Singapore). The animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanyang Technological University (Singapore). Details about synthesis and molecular characterization of PPCy-TPA and the preparation of PPCy-TPA NPs were reported in a previous publication.26 2.2 Equipment The particle size was determined by dynamic light scattering (DLS) (BI-200SM, Brookhaven, USA) with a detection angle at 90o. Fluorescence spectra were recorded using a fluorescence spectrometer (LS-55, Perkim Elmer, Waltham, MA, USA), and absorption spectra were measured using a UV-Visible spectrophotometer (Cary 4000, Varian, Palo Alto, CA, USA). NIR fluorescence images of animals were taken by a commercial IVIS spectrum in vivo imaging system (Caliper Life Sciences, Waltham, MA, United States). The exposure time was set at 0.2 seconds and the central wavelengths of the excitation and emission filters were set as 745 nm and 840 nm, respectively. 2.3 Optical setup for phantom imaging The optical setup of the NIR fluorescence imaging system for nanoparticle characterization is illustrated in Scheme S1. The infrared diode laser with a 785-nm central wavelength (FC-D-785, CNI, Changchun, Jilin, China) served as the excitation light source. A convex lens with a focal length of 7.5 cm was utilized for beam collimation, and a laser line filter (LL01-785, Semrock, Rochester, NY, USA) was used to reject undesired side bands. The diameter of the beam incident on the mice skin was adjusted to around 1.4 cm, and accordingly the maximum output power


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intensity was 0.248 W/cm2. The excitation and emission beams were separated by a dichroic mirror (LPD02-785RU, Semrock, Rochester, NY, USA). A long pass filter (BLP01-785R-25, Semrock, Rochester, NY, USA) was placed at the emission light path to suppress the excitation light. The fluorescence signal was acquired by a water-cooled SWIR high resolution InGaAs camera (SWIR VGA, Photonics Science, Robertsbridge, East Sussex, UK) with the assistance of a camera lens (GMHR3D25018C, Goyo Optical, Asaka, Saitama, Japan). The distance between the camera lens and the sample was set to 46 cm to obtain a 10-time demagnification. The two-layered skin phantom study was carried out to quantitatively characterize the fluorescence efficiency and penetration depth when using PPCy-TPA NPs as the contrast agent for fluorescence imaging. The top layer was to mimic the epidermal layer of the skin, which was made by mixing 0.045 g of agarose (PC0701-500G, Vivantis, Subang Jaya, Malaysia) and 1.62 mL of 2.5% (weight/volume) polystyrene microspheres (1 mm, Catalog No. 07310, Polysciences, Warrington, PA, USA) in 3-mL water, which yielded a reduced scattering coefficient of the skin phantom as 3.1 mm-1 at 785 nm representative of the average value of human epidermis and dermis 27. The bottom layer was made of PPCy-TPA solution embedded in 1.5% (weight/volume) agarose at a range of concentrations. The phantom study was conducted in two individual steps by altering the thickness of the top layer and the concentration of the PPCy-TPA NPs in the bottom layer, respectively. In each step the corresponding images of the two-layered phantom were recorded by the optical setup illustrated Figure S1. The fluorescence images of all phantoms were taken by the customized NIR fluorescence imaging setup shown in Figure S1. 2.4 In vivo biodistribution analysis.


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The HepG2 cell line (107 cells in 100 µL PBS) was injected subcutaneously into right back to generate tumor model. Each mouse used in biodistribution studies had a tumor volume of 100200 mm3, and was injected intravenously with 0.15 mL of PPCy-TPA NPs (700 mg/mL). At day 2 and day 7 post injection, mice were sacrificed after fluorescence imaging, and organs were taken out to acquire the fluorescence images. 2.5 In vivo photothermal treatment. HepG2 tumors were grown in the same way described above. Mice were injected intravenously with 0.15 mL of PPCy-TPA NPs (700 mg/mL). At 24 hours post injection, animals were anesthetized with 2% isoflurane in 100% oxygen. The entire tumor region was exposed to 808 nm continuous laser at power density of 1.77 W/cm2 for 10 minutes. Fluorescence images were taken every day with the same imaging parameters after photothermal treatment. 3. Results and Discussion 3.1 Preparation and characterization of PPCy-TPA colloidal nanoparticles Scheme 1a shows the chemical structure of PPCy-TPA, a donor-acceptor-donor triad. PPCyTPA itself is intrinsically hydrophobic and shows good solubility in nonpolar to midpolar organic solvents such as chloroform. In order to disperse PPCy-TPA into water, we employed a method of nanoprecipitation using an amphiphilic triblock copolymer Pluronic 127 (F127) as the stabilizer. Specifically, 5 mL of deionized water was quickly injected into the 1 mL mixture of PPCy-TPA (5 mg) and F 127 (69 mg) in THF under sonication. After 10 min sonication, THF was removed by dialysis against water over two days, resulting in PEGylated colloidal core-shell nanoparticles (NPs, Scheme 1a) with an average hydrodynamic diameter of 50 nm as measured


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by dynamic light scattering. Under TEM, the NPs were observed as non-uniform nanospheres which shrank to 27 nm in dry state under high vacuum. More details about the structural characterization of these NPs were reported in a previous publication.26 The present study mainly focuses on the application of these NPs for simultaneous fluorescence bioimaging and photothermal therapy at both in vitro and in vivo levels. Figure 1 shows the absorption and emission spectra of PPCy-TPA NPs in water. One can see two main absorption bands with the absorbance peaks located at 714 and 750 nm, respectively, accompanied by a small shoulder peak at 650 nm. PPCy-TPA NPs show a monomodal symmetric emission band centered at 810 nm, with a fluorescence quantum yield (Φ) of 0.2% (using pristine PPCy in chloroform (Φ = 46%) as the standard)26, 28. The low fluorescence quantum yield of PPCy-TPA NPs implies substantial non-irradiative pathways of the chromophores after light excitation. To examine this hypothesis, we characterized the photothermal effect of these NPs under continuous laser irradiation at 808 nm and a power


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Figure 1. Normalized UV-vis-NIR absorption (black) and fluorescence emission (red, λex= 650 nm) spectra of PPCy-DPA NPs dispersed in water. density of 1.77 W/cm2. Figure 2a shows dramatic photothermal effect of PPCy-TPA NPs at 25 mg/mL. The temperature of the NP dispersion increased from 27 to 53 oC after 10-minute laser irradiation and reached 59 oC when the concentration of PPCy-TPA NPs was doubled to 50 µg/mL. The photothermal conversion efficiency was calculated to be around 40%, which is higher than that of gold nanomaterials and comparable to that of narrow bandgap benzobisthiadiazole (BBT) derivatives.20-21, 29 To further investigate the photostability of PPCy-TPA NPs, we treated the NPs by 6 cycles of laser ON/OFF with the same wavelength and power as stated above. Specifically, after the dispersion of NPs (50 µg mL-1) was irradiated with NIR laser for 10 minutes (laser “ON”, Figure 2b), it was allowed to cool down naturally (without laser irradiation) to room temperature over 10 minutes (laser “OFF”). This cycle was repeated 5 times in order to investigate the


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photostability of PPCy-TPA NPs. The recorded temperature indicated no significant photoinduced degradation of PPCy-TPA NPs under the present experimental conditions (Figure 2b). The robust photostability of PPCy-TPA NPs observed here is considered as good as that of inorganic photothermal agents,30-31 so it is suitable for multiple treatment of long-term clinical use. 3.2 PPCy-TPA NPs for photothermal treatment of cancer cells In this section, we investigate the photothermally induced cytotoxicity of PPCy-TPA NPs against HeLa cells (human cervical carcinoma cell lines). Specifically, HeLa cells were incubated with 25 µg/mL of PPCy-TPA NPs for 6 hours. After irradiation with the same laser for 20 minutes, live/dead cells were differentiated by co-staining with calcein AM (green fluorescence for live cells) and propidium iodide (PI) (red fluorescence for dead cells) after photothermal treatment (Figure 2c and 2d). All cells in laser irradiation area were killed after the joint treatments of PPCy-TPA NPs and laser irradiation. In Figure 2d, the boundary (dashed line) of the laser spot can be observed, in which the dead cells within the laser spot show intense homogeneous red fluorescence. The cells outside the boundary of the laser spot showed strong green fluorescence, indicating that these cells without laser irradiation were alive. For a more quantitative evaluation of the photothermal cytotoxicity of PPCy-TPA NPs, HeLa cells subjected to the NIR laser irradiation over various periods were stained by PrestoBlue® reagent. In the experimental group, cells incubated with PPCy-TPA NPs for 6 hours in a 96-well plate was exposed to the 808-nm laser irradiation for 10 minutes. At this time point,


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Figure 2. (a) Temperature change plots of different concentrations of PPCy-TPA NPs in 2 mL water upon 10-minute irradiation by an 808-nm laser with a power density of 1.77 W/cm2. (b) Temperature elevation of PPCy-TPA NPs over 6 cycles of ON/OFF NIR laser irradiation. All the concentrations of the PPCy-TPA NPs are calculated based on the mass of PPCy-TPA without inclusion of the stabilizer. (c-d) Fluorescence images of HeLa cells co-stained by calcein AM/PI after incubation for 6 hours with PPCy-TPA NPs (25 µg mL-1), followed by irradiation (d) with the same laser for 20 minutes or without laser irradiation (c). (e) Viabilities of HeLa cells after incubation for 6 hours with PPCy-TPA NPs (25 µg mL-1), followed by the treatment with laser irradiation for 10 and 20 minutes, respectively. Cell viability was normalized to the control group without any treatment. Error bars indicate the standard deviations of five parallel samples. (f) Viability of HeLa cells after being incubated with various concentrations of PPCy-TPA NPs for 24 hours tested by PrestoBlue® reagent without laser irradiation.


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there was no apparent change in the viability of the two control groups (Figure 2e, red and green columns). However, the experimental group (Figure 2e, blue column) at 10 minutes showed about 40% dead cells as compared to the control groups (Figure 2e). Although Figure 2a shows significant photothermal effect in the aqueous dispersion of PPCy-TPA NPs at 25 µg/mL, the relatively low photothermally-induced cytotoxicity observed here after 10 minutes of laser irradiation could be attributed to the fact that not all of the NPs were internalized by the cells during the certain incubation period, while those NPs not internalized by the cells were washed away. As a consequence, the effective concentration of the NPs in the cells could be lower than 25 µg/mL, though further quantitative characterization is needed in the future. When the irradiation time was increased to 20 minutes, nearly 100% cell death was observed in the experimental group (Figure 2e). Biocompatibility of PPCy-TPA NPs was examined by incubating HeLa cells with dispersions of the NPs with a series of concentrations for 24 hours without any treatment of laser irradiation. Figure 2f shows the concentration dependence of cytotoxicity of NPs against HeLa cells. Obviously, PPCy-TPA NPs show minimal toxicity to HeLa cells without NIR laser irradiation, suggesting their good biocompatibility. 3.3 Imaging of PPCy-TPA nanoparticles in skin-like turbid phantoms The two-layered phantoms had two controlled variables, i.e. the thickness of the top layer and the concentration of PPCy-TPA in the bottom layer, to examine the maximum imaging depth using PPCy-TPA NPs with different concentrations as the contrast agent. The thickness of the top skin layer was increased from 0.5 to 2.5 mm with an increment of 0.5 mm while the concentration of the PPCy-TPA NPs was decreased from 10 to 1 then to 0.1 mg/mL. The bottom


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layer was made in a triangular shape and overlaid by the top layer, thus the imaging contrast of the PPCy-TPA NPs could be directly quantified as the ratio of the intensity of the triangular region to that outside the triangular region in a resulting fluorescence image. The representative fluorescence images acquired for different PPCy-TPA concentrations and the top layer thicknesses are illustrated in Figure 3. The exposure time of the camera was set to 20 ms to ensure image quality. From the left to right column in every row of Figure 3, the acquired images corresponded to a range of the top layer thickness from 0 mm to 2.5 mm with an increment of 0.5 mm while the concentration of PPCy-TPA NPs and the exposure time were fixed at a value indicated in the row header. Figure 3 shows that the outline of the bottom PPCY-TPA layer can be well recognized when its concentration and the exposure time were 10 mg/mL and 20 ms, respectively, even if the top layer thickness, i.e. the depth of the bottom layer, was increased to 2.5 mm as shown in Figure 3(f). The imaging depth decreased when the concentration was reduced from 10 mg/mL (row 1) to 1 mg/mL (row 2) and then to 0.1 mg/mL (row 3). Fluorescence images have been heavily distorted by strong light scattering induced by the top scattering layer when the top layer thickness was greater than 2 mm (Figure 3(l)) for a 1 mg/mL concentration or greater than 1 mm (Figure 3(p)) for a 0.1 mg/mL concentration. As expected, the measured fluorescence intensities could be improved by increasing the exposure time, for example from 20 to 100 ms, as shown in Figures 3(s)-(x), while the concentration of PPCy-TPA NPs remained at 0.1 mg/mL. In order to quantify the imaging contrast performance of PPCy-TPA NPs with concentrations of 1 mg/mL and 0.1 mg/mL at different target depths, the measured average fluorescence intensity and imaging contrast of the triangular bottom layer with PPCy-TPA as a function of the top layer thickness are plotted in Figure 4. Note that the imaging contrast is calculated as the


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ratio of the fluorescence intensity measured in the triangular region to that measured in the nontriangular region after dark background subtraction. It can be clearly observed from Figure 4 that both the fluorescence intensity and imaging contrast of the two concentrations decreased with the increase of the top layer thickness. Note that the PPCy-TPA layer with a concentration of 1 mg/mL exhibits a rapid drop in both fluorescence intensity and imaging contrast when the top layer thickness is increased from 0.5 mm to 1.5 mm. In contrast, the PPCy-TPA layer with a concentration of 0.1 mg/mL shows a much slower decrease. It is observed that the image of an object with a contrast below 2 is visually unrecognizable from background, which can be appreciated in Figure 3.


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Figure 3. Fluorescence images of triangular agar phantoms with PPCy-TPA NPs at the concentrations of 10, 1 and 0.1 µg/mL when the thickness of top layer was altered from 0 mm to 2.5 mm. (a)-(f) Fluorescence images of PPCy-TPA at a concentration of 10 µg/mL when the exposure time was 20 ms; (g)-(l) fluorescence images of PPCy-TPA at a concentration of 1 µg/ml when the exposure time was 20 ms; (m)-(r) fluorescence images of PPCy-TPA at a concentration of 0.1 µg/mL when the exposure time was 20 ms; (s)-(x) fluorescence images of PPCy-TPA at a concentration of 0.1 µg/mL when the exposure time was 100 ms.

Figure 4. (a) Measured average fluorescence intensity and (b) imaging contrast of the triangular bottom layer with PPCY as a function of the top layer thickness. The phantom results demonstrate that the fluorescence image of the bottom PPCy-TPA layer cannot be differentiated well from the background if the thickness of the top layer is larger than l mm and PPCy-TPA concentration is below or equal to 0.1 mg/mL, assuming an exposure time of 20 ms. In consideration of the fact that the thickness of the mouse skin (including the epidermis, dermis and hypodermis) is around 1 mm,4 the amount of injected PPCY needs to be higher than 2.2 mg to reach the minimum concentration (0.1 mg/mL) in mouse body. The phantom study was used to guide in vivo imaging by offering information on suitable PPCy-TPA concentrations to achieve reasonable imaging quality.


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3.4 In vivo and Ex vivo imaging in nude mice

Figure 5. In vivo images of back tumor model by HepG2 cell lines within 7 days (a-i) after tail injection of PPCy-TPA NPs. The amount of injected NPs was 0.15 mL of dispersion with a concentration of 0.7 mg/mL. (a) The mouse with the xenografted tumour (outlined with a black circle) under ambient light. (b-i) The mouse under NIR light irradiation with an excitation wavelength of 745 nm, an emission band pass filter of 840 ±10 nm, and the exposure time of 0.2 second.


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We used an in vivo tumor model generated by HepG2 or HeLa cell line to study the biodistribution of PPCy-TPA NPs in nude mice. An aliquot (100 µL) of PPCy-TPA NPs (0.7 mg/mL) was injected intravenously into a nude mouse with a tumor volume of 100-200 mm3 (Figure 5). The fluorescence images of the tumor mice acquired by the commercial IVIS system after injection of the NPs are illustrated in Figure 5 and Figures S1-3, respectively. While the distribution of the NPs in the tumor site can be easily imaged in vivo from the side position of the mice, only those NPs inside the liver can be clearly seen in the supine position. As most important organs, including kidneys, spleen and stomach, in mice are covered by the skin and partially overlap with the intestine after projection, and other vital organs (such as heart and lungs) are covered by the sternum, the in vivo imaging of nude mice organs is always challenging. In most situations only, the liver can be clearly seen as this large organ is just below the skin surface. Figure 5a shows that in the initial 2 hours the few PPCy-TPA NPs started to accumulate in the tumor site after the injection of PPCy-TPA NPs. The more accumulation of the NPs started to be clearly observed in both liver and tumor from 10 hours be identified clearly in day 2 and 3 (Figure S1). Importantly, the outlines of liver and spleen can be observed when the circulation time was extended to 2-5 days (Figure 5f-h). No significant decrease of fluorescence intensity was observed during 5-7 days, which demonstrates that the longer blood circulation time than that of previous reported NIR agents26 ensures the higher effective concentration of PPCy-TPA NPs in body. Not only in HepG2 tumor tissues, the PPCy-TPA NPs can also accumulate in tumors generated by HeLa cells, which shows similar circulation fate of NPs in Figure S2-S4. These in vivo


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fluorescence images help us determine the accurate accumulated amount of the PPCy-TPA NPs in the key organs, which can ensure the treatment time and efficiency. We carried out a more quantitative measurement of the biodistributions of the PPCy-TPA NPs by imaging the anatomy organs tissues ex vivo from the sacrificed mice body on Day 2 and Day 7 after injection, respectively. The acquired ex vivo fluorescence images are shown in

Figure 6. (a-b) Ex vivo fluorescence images of the anatomical organs from tumor mice (HepG2), which were sacrificed at 2 days (a) and 7days post injection (b) of PPCy-TPA NPs. (c) Profile of fluorescence intensities reflecting the distribution of the NPs in different organs.


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Figure 6, in which the spleen and the liver show the strongest fluorescence intensities, while other organs have much lower emission or nearly no emission, which agrees well with the in vivo results in Figure 5. Such significant uptake by mononuclear phagocytic system (MPS) including liver and spleen has been widely observed in various NPs with sizes in the range of 20-500 nm.32-36 While the NPs on Day 2 post injection showed a moderate extent of accumulation at the tumour site compared to other organs, their accumulation at the tumour site was significantly enhanced on Day 7 post injection. Moreover, the average fluorescence intensity (counts/cm2) of each organ is calculated based on the mean value in the organ area, which are plotted as histograms in Figure 6c, from which the relative emission intensities of the organs are quantified. These results suggest that PPCy-TPA NPs accumulate primarily in spleen and liver after 24 hours following the tail-vain injection of the NPs. 3.5 In vivo photothermal therapy on tumor mice We next investigated the potential of PPCy-TPA NPs for photothermal therapy of a tumormodel in mice. An aliquot (150 µL) of PPCy-TPA NPs (0.7 mg/mL) was intravenously injected into mouse with a tumor volume of 100-200 mm3, followed by a laser irradiation treatment after 24 h since the injection. The strong fluorescence from the tumor site could be observed 1-day post-injection (Figure 7a), which suggests most of the NPs were accumulated in tumor tissues by EPR effect. After laser irradiation, the color of skin on tumor site turned to white, presumably caused by laser heating inside the tumor tissue, which turned into brown blood crust in the next two weeks (Figure 7b). One can see that the fluorescence intensity of PPCy-TPA NPs accumulated in tumor sites decreased significantly after laser irradiation treatment, while the fluorescence intensity was partially recovered as a result of accumulation of recovering tissues at the blood crust (Figure 7a). The increasing of the fluorescence intensity might be due to the


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dissociation of tumor tissue after photothermal treatment, as the tissue dissociation could be easier for NPs to accumulate again37. Fortunately, the blood crust disappeared after 18 days

(Figure 7b) and the mice were recovered without any weight loss (Figure 7d). In contrast, the tumor without laser treatment grew quickly with a final tumor volume of 600-700 mm3 (Figure 7b).


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Figure 7. (a) The fluorescence images of tumor mice generated by HepG2 cell lines intravenously administrated with aqueous suspensions of PPCy-TPA NPs on day 1 post-injection mice were treated by 808 nm laser irradiation at 1.77 W/cm2 for 10 minutes. (b) The images of tumor mice treated with and without laser irradiation within 18 days. (c) The volume size of tumor tissue with and without treatment was calculated using the formula Length × Width2/2. (d) The weight changes of three experimental groups with treatment and control group without treatment with PPCy-TPA NPs and the laser irradiation. G1-4 represent 4 groups of mice, in which G1-3 were treated with the NPs and the laser irradiation while G4 without such treatment. 4. Conclusion We have presented a new type of theranostic colloidal NPs composed of PPCy-TPA chromophores with strong light absorption and fluorescence in the NIR optical window. These NPs exhibit excellent optical properties in imaging and high photothermal conversion with robust photostability. Moreover, significant death of HeLa cells was observed due to the hyperthermal effect. The PPCy-TPA NPs also showed effective tumor-specific accumulation after intravenous injection. More importantly, the PPCy-TPA NPs could eliminate the tumor safely following photothermal treatment. These results demonstrate that the PPCy-based NPs are promising theranostic agents for simutaneous cancer imaging and therapy. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Schematic of the home-built near-infrared fluorescence imaging setup, In vivo distribution of PPCy-TPA NPs, In vivo images of back tumor model and Ex vivo fluorescence images of the anatomical organs from tumor mice. (PDF) ACKNOWLEDGEMENTS


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M.W. is grateful to the funding support by a start-up grant (M4080992.120) of Nanyang Assistant Professorship from Nanyang Technological University and AcRF Tier 2 (ARC 36/13) and AcRF Tier 1 (RG 20/16) from the Ministry of Education, Singapore. S.H. gratefully acknowledges the Ph.D. research scholarship from Nanyang Technological University. AUTHOR INFORMATION Corresponding Author * Mingfeng Wang. E-mail: [email protected] * Liu Quan. Email: [email protected] Present Addresses W. Liu: currently in the Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. REFERENCES 1. Fabian, J.; Nakazumi, H.; Matsuoka, M., Near-Infrared Absorbing Dyes. Chem. Rev. 1992, 92, 1197-1226. 2. Guo, Z.; Park, S.; Yoon, J.; Shin, I., Recent Progress in the Development of near-Infrared Fluorescent Probes for Bioimaging Applications. Chem. Soc. Rev. 2014, 43, 16-29. 3. Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z., Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. 4. Kievit, F. M.; Zhang, M., Cancer Nanotheranostics: Improving Imaging and Therapy by Targeted Delivery across Biological Barriers. Adv. Mater. 2011, 23, H217-H247. 5. Melancon, M. P.; Zhou, M.; Li, C., Cancer Theranostics with near-Infrared LightActivatable Multimodal Nanoparticles. Acc. Chem. Res. 2011, 44, 947-956.


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