High-Performance Identification of Human Bladder Cancer Using

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Biological and Medical Applications of Materials and Interfaces

High-Performance Identification of Human Bladder Cancer Using Signal Self-Amplifiable Photoacoustic Nanoprobe Di Zhang, Ziqi Wang, Lu Wang, Zhichao Wang, Hongzhi Wang, Guangbin Li, Zeng-Ying Qiao, Wanhai Xu, and Hao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08357 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Applied Materials & Interfaces

High-Performance Identification of Human Bladder Cancer Using Signal Self-Amplifiable Photoacoustic Nanoprobe Di Zhang, †,§ Ziqi Wang, ‡,§ Lu Wang, ‡ Zhichao Wang, ‡ Hongzhi Wang, ‡ Guangbin Li, ‡ ZengYing Qiao, †,* Wanhai Xu, ‡,* and Hao Wang†,* †

CAS Center for Excellence in Nanoscience, Laboratory for Biomedical Effects of

Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing, 100190, China ‡

Department of Urology, the Fourth Hospital of Harbin Medical University, Heilongjiang Key

Laboratory of Scientific Research in Urology, Harbin, 150001, China. KEYWORDS: Self-assembly, Photoacoustic, Nanoprobe, Cancer, Bioimaging

ACS Paragon Plus Environment

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ABSTRACT: Cancer diagnostics has been an important research field and identification of small lesions that are less noticeable plays a vital role in thoroughly removing the tumor, thereby reducing the recurrence rate of cancer. Herein, we synthesized a signal self-amplifiable photoacoustic liposomal nanoprobe composed by ammonium hydrogen carbonate (AHC) payload and aggregated purpurin-18 (P18) within the bilayer. Under PA laser irradiation, P18 aggregates efficiently generated local heat, leading to the launch of wide-band ultrasonic emission. In parallel, the heat also triggered the decomposition of AHC and production of CO2 bubbles, which consequently dramatically amplified the acoustic signal. For clinical translation, by decorating bladder cancer specific CD44v6 antibody onto nanoprobe, we were capable of utilizing this high sensitive and specific PA probe for human bladder cancer tissue imaging. The results indicated that small tumor lesion (< 5 mm) was identified and the tumor-to-normal tissue (T/N) ratio was ~18 folds enhancement by using this PA probe, which rendered the tumor boundary distinct. All together, we developed a new strategy for exploring high-performance imaging probes which might potentially benefit for the imaging-guided surgery in clinic.


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INTRODUCTION Bladder cancer (BC) is a common malignancy in urinary system and often exists in multiple forms. The recurrence rate of BC can reach up to 78% at 5 years, which is the highest among all kinds of cancers.1-2 Traditional imaging methods, like ultrasound and computed tomography (CT), have reduced diagnostic rates when the tumor size is smaller than 10 mm.3-4 White light cystoscope, a central method for the diagnosis, resection, and surveillance of BC, is limited to identify the flat tumors and has challenge to identify the tumor boundary.5 These limitations lead to missed or incomplete resection during surgery, thereby the risk of recurrence increased. Therefore, the exploration of a new method to precisely identify tumor and its boundary has a practical significance in reducing the recurrence rate of bladder cancer. In recent years, photoacoustic (PA) imaging technique has emerged as a promising biomedical diagnostic tool due to its high spatial resolution and deep tissue penetration capabilities.6-9 The PA effect is based on acoustic wave generation induced by the transient thermoelastic expansion of optical excitation. There are various inorganic/organic nanoparticles used as contrast agents of PA imaging, such as gold nanoparticles,10-12 carbon nanotubes,13-15 and other complexes materials.16-19 These materials have a relatively high ability to absorb the energy of the near-infrared (NIR) light and convert it into heat. In contrast to inorganic materials, organic nanoparticles such as semiconducting polymer nanoparticles have reported widely.20-23 Considering the clinical applications, liposomes with good biocompatibilities have been developed. However, because they had no intrinsic absorption in NIR region, NIR dyes had to be embedded into these nanoparticles for sensitive visualization of the region of interests (ROI) by PA imaging.24-25 For example, porphyrin conjugated with


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phospholipid self-assembled into liposome-like structure, which was investigated as biophotonic contrast agents with the high optical-thermal conversion efficiency for PA imaging.26 Purpurin18 (P18), a porphyrin derivative, has strong absorption at NIR region which is generally utilized as photosensitizer for tumor PA imaging27-28 or photodynamic/photothermal therapy.29 In our previous work, an enzyme-responsive peptide-P18 self-assembled into nanofibers in tumor site where assembly induced retention (AIR) effect exhibited, resulting in improved PA imaging signal and tumor therapeutic efficacy.29 NIR dye squaraine was also nano-confined in liposomes as PA imaging contrast agent, which showed good sensitivity, high spatial resolution and deep tissue penetration in ROI areas.30 However, there is a bottleneck in developing the PA contrast agent because the pursuit of resorting to high optical-thermal conversion to enhance PA signal may lead to thermal-toxicity. Therefore, a new strategy needs to be developed for enhancing the PA signal in vivo. One of the possible solutions is to amplify the ultrasonic signal generated by thermoelastic expansion.31 It is well known that bubbles have a high degree of echogenicity, which could be used as an object to reflect the ultrasound waves and enhance the ultrasound backscatter, leading to the amplification of acoustic signal.32-34 Herein, we demonstrate a sensitive and specific liposomal nanoprobe and successfully utilize it for human bladder cancer tissue photoacoustic imaging for the first time, to the best of knowledge (Scheme 1). Nanoconfined P18 aggregates and ammonium hydrogen carbonate (NH4HCO3, AHC) were encapsulated into liposome simultaneously to obtain supramolecular assemblies P18/AHC⊂ ⊂L. P18 molecules were adjusted into aggregates in the hydrophobic bilayers of liposomes by changing the mixing ratios of P18 and phospholipid. The fluorescence quenching of P18 aggregates led to thermal release after NIR laser irradiation of PA instrument, which resulted in the transient thermoelastic expansion and launch of ultrasonic signal.


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Meanwhile, the heat converted by P18 aggregates could trigger the thermal reaction of slow decomposition of AHC followed by lasting generation of CO2 bubbles at temperature around 42 o

C.35 The high difference in acoustic impedance between the bubbles and the surrounding tissues

makes bubble highly reflective, resulting in the enhanced acoustic backscattering from surrounding tissues. The controllable generation of bubbles specially realized the spatially controllable amplification of PA signal in region of interest (ROI) in vivo. In human bladder Scheme 1. a) Nanobubbles generation in P18/AHC⊂ ⊂L under laser irradiation. b) High-performance identification of human bladder cancer and tumor boundary using P18/AHC⊂ ⊂L as PA imaging probes.


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cancer tissue, by decorating bladder cancer specific CD44v6 antibody onto nanoprobe, CD44v6P18/AHC⊂ ⊂L generates bubbles under laser irradiation and magnifies tumor-to-normal tissue ratio (T/N) of PA signal up to 18 folds, and hence the tumor boundary was distinguished clearly. Meanwhile, because few decomposition of AHC occurred under irradiation interval, the stable and persistent PA signal amplification can be obtained, offering the feasibility for long-time imaging-guided surgery in clinic. EXPERIMENTAL SECTION Materials. Purpurin-18 (P18), agarose, uranium acetate and diaminobenzidine were purchased from Sigma-Aldrich. L-α-Phosphatidylcholine (PC), DSPE-PEG2000, DSPEPEG2000-NHS and cholesterol (CH) were purchased from Avanti Polar Lipids. CD44v6 and IgG antibody were purchased from Abcam and Protentech Co., respectively. EJ human bladder and MCF-7 tumor cell lines were purchased from cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Female BALB/c mice (6-8 weeks, 17-18 g) were purchased from Vital River laboratory animal technology Co., Ltd. (Beijing, China). Preparation of Supramolecular Assembly. The nano-confined the aggregates of P18 and AHC loaded liposomes were prepared through adjusting the concentration of P18 and AHC. Briefly, different concentration of P18 was added into the ethanol solution of L-αPhosphatidylcholine (PC, 3.04 mg) and cholesterol (CH, 0.76 mg). P18/AHC⊂ ⊂L were formed by rapidly injecting the ethanol solution into PBS (1 mL) containing AHC with different concentrations, followed by stirring for 2 h. Finally, the ethanol in the liposome solution was evaporated with rotatory evaporator. P18/AHC⊂ ⊂L was prepared with the similar procedure. And


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the UV/Vis absorption, fluorescent intensity and PA signal intensity of P18/AHC⊂ ⊂L in PBS were investigated with the same methods of P18 (see Supporting Information). Characterization of P18/AHC⊂ ⊂L. The morphology and size of P18/AHC⊂ ⊂L were studied by transmission electron microscope (TEM, Tecnai G2 20 S-TWIN) with an accelerating voltage of 200 KeV. The hydrodynamic diameter of liposomes were measured by a dynamic light scattering (DLS) analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd). Synthesis of CD44v6-Probe and IgG-Probe. The concentration of CD44v6 antibody and IgG antibody were 1 mg/mL. P18⊂L and P18/AHC⊂L were synthesized as previous described.[3] We mix antibody and nanoparticle in different groups (molar ratio of antibody: PC=1:100) and the resultant mixture was stirred overnight at 4 oC to obtain the CD44v6-Probe (CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L) and IgG-Probe (IgG-P18⊂ ⊂L and IgG-P18/AHC⊂ ⊂L). PA Imaging of Bladder Tumor in Mice. The PA imaging was performed following the procedure of MCF-7 tumor model as above mentioned (see Supporting Information). CD44v6P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L (100 µM, 200 µL) dispersed into PBS was intravenous injected into mice bearing EJ human bladder tumor through tail vein, respectively. After the injection, the mice were scanned with MSOT (mode: MSOT 128, excitation wavelength at 750 ±1 nm) at 4 h. H&E Staining. Human bladder tissues were immersed in 4% paraformaldehyde overnight at room temperature and then transferred to 60% ethanol. After dehydrated through a serial alcohol gradient (60%~100%) for each 4 h, the tissues were immersed in xylene twice for 0.5 h. Finally, the samples were embedded in paraffin wax pre-heating at 60 oC for 2 h, then slicing up


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to 5-um-thick sections and drying at 80 oC overnight. Before immunostaining, tissue sections were dewaxed in xylene 3 times for each 10 min and then rehydrated through decreasing concentrations of ethanol (100% ~ 80%) for each 5 min, followed by washing in distilled water. Then the tissue sections were stained with hematoxylin and eosin (H&E). After staining, sections were dehydrated through increasing concentrations of ethanol (70% ~ 100%) and xylene. Immunohistochemistry. Tissue sections and dehydrated process were performed as previous described. Then antigens were retrieved in 10 mmol/L citrate buffer (pH 6.0), heating in a pressure cooker for 20 min followed by cooling at room temperature and washing by PBS for 3 times. Next, 50 % bovine normal serum was used for protein blocking for 1 h, further incubated with 5 ug/ml anti-CD44v6 monoclonal mouse IgG antibody overnight at 4 oC. Next day, sections were washed 3 times by PBS and incubated with the appropriate biotin-conjugated secondary antibody subsequently in streptavidin solution, and color development was performed using 3,3diaminobenzidinetetrahydrochloride (Vector Laboratories) as a chromogen. Finally, the sections were counterstained with Harris’s haematoxylin for 5 s, which was then dehydrated and mounted also as previous described. PA Imaging of Human Bladder Tissue. The bladder nomal tissue and tumor tissue were treated with CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L overnight at 4 oC, respectively. Then the tissues were immersed PBS for 2 h, followed by washing with PBS for three times. Finally, the tissues were scanned with MSOT (mode: MSOT 128, excitation wavelength at 750 ±1 nm). RESULTS AND DISCUSSION Construction and Properties of PA Nanoprobe. P18/AHC⊂ ⊂L was prepared using an ethanol injection method, with P18 in the hydrophobic bilayer and AHC in hydrophilic interior


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(Scheme 1). Dynamic light scattering (DLS) results showed that hydrodynamic diameter of P18/AHC⊂ ⊂L was 88 ± 5 nm with narrow size distribution (polydispersity index: 0.16) and the liposomes kept stable up to 12 h (Figure 1a). The uniform vesicular structures of P18/AHC⊂ ⊂L with a size of 81 ± 12 nm were also observed by transmission electron microscope (TEM) (Figure 1a, inset). Moreover, the aggregation behaviors of P18 played an important role in intensity and sensitivity of PA imaging, and we investigated the UV-Vis absorption and fluorescence spectra of P18 in the mixing solvent with different ratios of DMSO and H2O (Figure S1). The formation of P18 aggregates was verified due to the appearance of absorption peak at ~760 nm (Figure S1b) and the quench of fluorescence signal at ~720 nm (Figure S1c). Meanwhile, the aggregation of P18 resulted in the obvious increase of PA signal (Figure S1d), implying the aggregated P18 could be loaded into liposome for enhancing PA signal. The aggregation-induced enhancement of the photoacoustic signal could be attributed to the change of photothermal conversion capacity,36 and P18 aggregates showed ~19 folds enhancement of photothermal conversion efficiency than P18 monomers, which was calculated according to the heating/cooling curves and corresponding calculation equation in supporting information (Figure S2). Therefore, the weight ratios of P18 to liposome were changed from 0.25% to 10%, and aggregation of P18 was realized as the P18 concentration increase, which could be proved by UV-Vis and fluorescence spectra of P18/AHC⊂ ⊂L (Figure 1b and 1c). Another distinct feature for aggregates was an induced circular dichroism (CD) effect,37-38 occurring on chiral excitonic coupling of transition dipole moments, and hence CD spectra of P18/AHC⊂ ⊂L were studied. As shown in Figure 1d, two bands with opposite signs (exciton couplet) were observed as the concentration of P18 increase, indicating the chiral properties of the aggregates. P18 with loading content higher than 10% was also performed, but there were obvious aggregates and


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precipitates after the loading. Therefore, the P18/AHC⊂ ⊂L with P18 loading content of 10% was chosen as optimal supramolecular assemblies for further application.

Figure 1. a) Number size distribution of the P18/AHC⊂ ⊂L (1 mg/mL) measured by DLS in aqueous solution at 4 h and 12 h; Insert is TEM images of P18/AHC⊂ ⊂L (1 mg/mL) in aqueous solution. b) Normalized UV/vis absorption spectra changes, c) the corresponding fluorescence intensity (λex: 680 nm) changes and d) CD spectra of P18/AHC⊂ ⊂L with variable mixing ratios from 0.25% to 10% (P18/phospholipids). e) PA signal intensity of AHC ⊂L, P18⊂ ⊂L and P18/AHC⊂ ⊂L (P18/phospholipids: 10%). The values were expressed as mean ± SD (N = 3). f) PA signal changes of P18⊂ ⊂L and P18/AHC⊂ ⊂L at different time points in 10 h under continuous laser irradiation. P18 concentration: 10 µM.


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To investigate whether the PA signal of P18/AHC⊂ ⊂L was amplified by thermal triggered generation of bubbles, the solution of AHC⊂ ⊂L, P18⊂ ⊂L and P18/AHC⊂ ⊂L (Concentration of P18 was 5 µM) was injected into the agarose patterns and scanned using the multispectral optacoustic tomography, separately (MSOT, obtained at 750 ± 1 nm). The average PA signal intensities were recorded through mean pixel intensity at the same area, and the PA signal of P18/AHC⊂ ⊂L was approximately magnified 3 folds compared to that of P18⊂ ⊂L (Figure 1e). In addition, at the same concentration of AHC, PA signal enhanced with increased concentration of supramolecular assemblies (Figure S3), and the P18 concentration of 10 µM was selected for further study. The long-term enhanced PA signal of P18/AHC⊂ ⊂L was also observed in 10 h under the continuous laser irradiation, whereas the PA signal of P18⊂ ⊂L kept instant in the same observed time scale (Figure 1f). However, the PA signal of P18/AHC⊂ ⊂L increased sharply at the beginning of 1 h, then slowly decayed during the subsequent measurement and finally reached its stable state. Based on above experimental results, we speculated that the slow decomposition of AHC produced CO2 bubbles for 8 h, which caused the PA signal enhancement. After that, the AHC was run out and subsequently resulted in the decrease of PA signal. In practical, discontinued laser irradiation was required for long-term monitoring PA signal. We therefore observed the PA signal intensity for 12 h through laser irradiation on P18/AHC⊂ ⊂L every other hour. The results clearly showed that no remarkable decreased signal was observed at this condition, indicating the good stability of the nanoprobe (Figure S4). Mechanisms of Enhanced PA Signal. We attempted to elucidate the possible mechanisms of enhanced PA signal. First of all, the photothermal transduction of P18/AHC⊂ ⊂L was primarily studied under a laser irradiation for 1 min, and the time-dependent temperature changes of different solutions were monitored via thermal camera. As shown in Figures 2a and 2b, the


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concentration-dependent temperature increment was observed. Beside light-to-heat conversion effect of aggregated P18, we hypothesized that the heat would vaporize AHC and generate CO2 bubbles that remarkably augmented PA signal. In order to confirm this assumption and determine the temperature of CO2 bubbles generation, the hydrodynamic diameter and PA imaging of P18/AHC⊂ ⊂L was monitored at different temperatures from 35 oC to 44 oC (Figure S5). As the temperature increased up to 40 oC, the PA signal was enhanced and simultaneously the particle size became larger, suggesting that the generation of bubbles induced the disruption of liposomes and further aggregation. Meanwhile, the AHC (0.5 mM) and P18 aggregates (10 µM) mixtures were dissolved in H2O in the presence of FITC as a fluorescence indicator. As can be seen from Figure 2c, large amount of bubbles with green fluorescence were observed as the temperature increased to 42 oC under NIR laser irradiation, while no bubbles appeared in AHC or P18 water solution at the same condition, indicating that, upon laser irradiation, P18 aggregates effectively converted the light energy to heat that simultaneously decomposed AHC into CO2 bubbles. Meanwhile, we also tested the bubble generation capability of AHC and P18 aggregates mixtures at 37 oC and the results exhibited that few bubbles were observed after 10-h incubation (Figure 2c), suggesting that AHC barely decomposed at the physiological temperature. Finally, we carried out the TEM experiments to study the morphological features of P18/AHC⊂ ⊂L at different temperatures. Obviously, the liposome membrane was disrupted at 42 oC under NIR laser irradiation (Figure 2d). In a sharp contrast, intact and integrated membrane layer was ambiguously observed at 37 oC (Figure 2e). The morphology change of nanoprobe before and after NIR laser irradiation was verified by scanning electron microscope (SEM) (Figure S6), which was in accordance with TEM results. We deduced that the higher temperature caused decomposition of AHC and subsequent generation of CO2 bubbles and destroyed the integrity of


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membranes. Together, our data showed that the absorbed energy by P18 aggregates after irradiation converted into heat, which triggered the AHC decomposition for releasing CO2 bubbles, leading to amplified transient thermoelastic expansion and thus enhanced emission of the ultrasonic signal.

Figure 2. a) The image of device and the temperature increase images of P18/AHC⊂ ⊂L (P18 concentration: 10 µM and 20 µM). b) Time-dependent temperature increase curves with different P18 concentrations of 10 µM and 20 µM, titanium-doped sapphire femtosecond pulse laser, 750 nm, 150 mW, flare diameter: 4 mm). c) Photos of P18 aggregates, AHC and AHC/P18 aggregates in FITC aqueous solutions under NIR laser irradiation, and photos of AHC/P18 aggregates in FITC aqueous solutions at 37 o

C. TEM images of d) P18/AHC⊂ ⊂L after irradiation by NIR laser for 30 s and e) P18/AHC⊂ ⊂L at 37 oC.


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Deep Penetration and High Resolution of PA Nanoprobe. PA imaging exhibits excellent capability of deep tissue penetration and high spatial resolution compared to fluorescence imaging. Agarose pattern (2 % in water, w/w) was utilized to simulate the artificial tumor tissues. The PBS solution of P18/AHC⊂ ⊂L (20 µL) was injected into the agarose pattern with syringe (diameter of pinhead: 0.4 mm), and cross pattern was designed as shown in Figure 3a. The agarose was longitudinally scanned by MSOT at 750 ± 1 nm, and after reconstruction all slices

Figure 3. a) The schematic illustration of the agarose pattern utilized to investigate the deep penetration and the spatial resolution of the PA contrast agents of P18/AHC⊂ ⊂L. b) High spatial resolution 3D PA imaging of agarose pattern with cross pattern of P18/AHC⊂ ⊂L. c) the PA signal of P18/AHC⊂ ⊂L in cross section (Top) and in different depths (Bottom) of the agarose pattern scanned by MOST. The signal intensity in spatial distribution was indicted by pink arrow. d) The depth-dependent PA (Top) and fluorescence (Bottom) signals. e) The quantification of Signal/Noise (S/N) of PA and fluorescence signals at different depths.


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of cross sections, we could obtain high spatial resolution three-dimensional (3D) PA imaging through combining the 2D images, indicating that there was no significant attenuation in any direction (Figure 3b). The PA signal was quantified in longitudinal direction of pink arrow (Figure 3c). The NIR imaging was also studied in the same condition with P18 monomer as florescence probe. We observed that the PA signal kept almost constant in the 18-mm depth of agarose pattern, while NIR signal showed apparent attenuation even at 6-mm depth (Figure 3d and 3e). Furthermore, the PA imaging showed clear boundary between P18/AHC⊂ ⊂L solution and blank agarose in the diameter of 0.4 mm (Figure 3b and 3c), implying the efficient recognition of PA signal in small sample. All these results demonstrated that P18/AHC⊂ ⊂L could be applied as an optimal PA imaging contrast agent with deep penetration and high spatial resolution, which was important for enhancing T/N ratio of PA imaging in vivo. Sensitivity of PA Probe in Vivo. In order to verify the sensitivity of PA probe in vivo, female BALB/c nude mice were used as animal model and the MCF-7 tumor cells were transplanted subcutaneously into the right lateral hind hip of mice. After the tumor had developed, P18⊂ ⊂L, AHC⊂ ⊂L and P18/AHC⊂ ⊂L was intravenously injected into mouse, respectively. The mouse was scanned with a PA imaging instrument (MSOT) at 30 min to 6 h (Figure S7). Compared with P18⊂ ⊂L and AHC⊂ ⊂L, the P18/AHC⊂ ⊂L displayed evidently enhanced PA signal, implying that bubble generation was the key factor for PA signal amplification in the time scale. The PA signal of P18/AHC⊂ ⊂L was higher than that of P18⊂ ⊂L, implying the spatially controllable bubble generation of P18/AHC⊂ ⊂L in tumor sites. In addition, the T/N of tumor to surrounding tissues was significantly enhanced, which played a critical role in determining the sensitivity and specificity of the imaging technique. At the same concentration (200 µL, 100 µM), the T/N ratio of mice group treated by P18/AHC⊂ ⊂L and P18⊂ ⊂L was ~13 and ~3, respectively. Therefore, the


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tumor boundary was significantly clear in P18/AHC⊂ ⊂L treated groups, which could not be identified in P18⊂ ⊂L and AHC⊂ ⊂L groups. The biocompatibility of PA contrast agents was also important for their further application in vivo. Therefore, the cytotoxicity of P18/AHC⊂ ⊂L was investigated, and there was no significant toxicity at the concentration up to 2 mg/mL, even with the 1 min laser irradiation (Figure S8). Therefore, P18/AHC⊂ ⊂L can be safely used as the sensitive and long-time spatially controllable PA contrast agent for tumor imaging. For further application in BC imaging, CD44v6 antibody was linked onto P18/AHC⊂ ⊂L for targeted PA imaging in human bladder cancer xenografted mice (EJ cell line). It was well known that CD44v6 was a cell surface protein which contributed to cell migration, cell adhesion, tumor progression and metastasis.39 It was also identified as a prognostic factor in various of malignancy like colorectal and breast cancer, as well as bladder cancer.40-41 In BC, CD44v6 was significant over-expression compared to normal bladder tissue,42-43 and it was considered as a new avenue for BC imaging. To realize the specific imaging of tumor, we further coupled CD44v6 antibody onto P18⊂ ⊂L and P18/AHC⊂ ⊂L, to gain CD44v6-P18⊂ ⊂L and CD44v6P18/AHC⊂ ⊂L, respectively (Figure 4a). For verifying the efficient accumulation of CD44v6P18/AHC⊂ ⊂L into tumor site, the biodistribution of nanoprobes was investigated by fluorescent imaging of xenografted mice through incorporating a near-infrared (NIR) fluorescent probe squaraine dye into the CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L nanoparticles. The real-time biodistribution images of the nanoparticles in vivo and ex vivo at 4 h post-injection were observed (Figure S9). In both nanoparticle treated groups, the tumors exhibited stronger fluorescence than other harvested organs, i.e., heart, liver, spleen, kidney and lung, proving the effective accumulation of nanoprobe into tumor in mice. The accumulation amounts of both


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nanoparticles in tumor were similar through semi-quantitative calculation of fluorescent intensity (Figure 4b). However, CD44v6-P18/AHC⊂ ⊂L exhibited ~3.2 fold higher PA signal than that of CD44v6-P18⊂ ⊂L in tumor site, demonstrating that in situ bubble generation could amplify PA signal intensity (Figure 4c and 4d). The results were in accordance with that of MCF-7 tumor

Figure 4. a) The CD44v6 labeled process on P18/AHC⊂ ⊂L and its targeting on bladder tumor cells. b) Quantitative analysis for the biodistribution of nanoprobes in major organs. c) PA imaging of tumor in

EJ xenografted mice after treated by CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L for 4 h. The white dashed circles indicate the outlines of cross section (normal tissue) and tumor site of mice. White arrows indicate tumors. Scale bars, 3 mm. d) PA signal in bladder normal tissue and tumor. The values were expressed as mean ± SD (N = 3).


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model, proving the universality and reliability of the nanoprobes in vivo. Furthermore, the stability of nanoprobes was evaluated by detecting the change of plasma concentrations. After administration of CD44v6-P18 ⊂L and CD44v6-P18/AHC⊂ ⊂L in mice, the PA signals of plasma at different time points were measured. The half-lifes (T1/2) of both nanoparticles were similar, implying the good stability of CD44v6-P18/AHC⊂ ⊂L in vivo (Figure S10). Considering the further clinic application, the cytotoxicity of CD44v6-P18/AHC⊂ ⊂L was also evaluated in EJ cell line, showing no significant toxicity at various concentrations (Figure S11). The histopathology assays (H&E staining) of main organs in mice treated by targeted nanoprobes was used to evaluate the bio-safety, displaying no significant organ damage in vivo (Figure S12). High-Performance Imaging of Human Bladder Tumor. After demonstrating the sensitivity of CD44v6-P18/AHC⊂ ⊂L in animal model, we further applied this optimal PA probe in human bladder cancer imaging. First, in order to verify the differential expression of CD44v6, we collected normal and cancer tissues from 6 different patients with radical cystectomy (Figure 5). In normal bladder tissue, CD44v6 showed no immunoreactivity when staining to the paraffinembedded samples. Conversely, the intensity of immunostaining for CD44v6 was strong in bladder cancer tissue (Figure 5a and 5b). These results indicated that CD44v6 can be used as a credible target. Subsequently, to verify the detected PA signal on tumor lesions was attributed to cancer-specific binding of CD44v6, IgG antibody was also coupled onto liposomes (IgG-P18⊂ ⊂L and IgG-P18/AHC⊂ ⊂L) as control groups. Normal bladder tissues and tumor tissues from the same patient were treated with IgG-probe (IgG-P18⊂ ⊂L and IgG-P18/AHC⊂ ⊂L) and CD44v6probe (CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L) respectively, which were imaged with the PA imaging system. As we expected, after the treatment of IgG-probe, only weak PA signal was observed on normal and tumor tissue both identified with H&E staining. After incubated with


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CD44v6-probe, PA signal was detected on tumor tissue rather than normal tissue (Figure 5c). Importantly, the PA signal intensity of CD44v6-P18/AHC⊂ ⊂L is significantly higher than CD44v6-P18⊂ ⊂L in the small tumor tissue of ~5 mm (Figure 5d and S13), which indicated that CD44v6-P18/AHC⊂ ⊂L exhibited the effect of amplifying PA signal. Another five groups of normal-tumor tissue (< 5 mm) from different patients were also treated with both probes (Figure

Figure 5. a) Normal urothelial and tumor tissues that were obtained from patients with radical cystectomy b) Expression of CD44v6 in normal urothelium and bladder cancer tissue. Original magnification 40×. c) The PA imaging of normal urothelium and tumor tissue after treatment with IgG-probe (Top) and CD44v6-probe (Middle) and corresponding H&E images (Bottom), (N = 6). Scale bars, 3 mm. d) Corresponding expression scores in each group. The values were expressed as mean ± SD. Asterisks (*) denote the statistical significance: ***p < 0.001.


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S14), and the obvious enhancement of PA signal was observed in all the groups, further proving the reliability of the imaging probe for detecting small human tumor (< 5 mm). Efficient Identification of Tumor Boundary. For further demonstrating the improved spatial resolution of CD44v6-P18/AHC⊂ ⊂L, bladder tissue was obtained including tumor and normal tissue. Next, we divided the bladder tissue into two pieces and treated with CD44v6-P18⊂ ⊂L and CD44v6-P18/AHC⊂ ⊂L respectively. Compared with CD44v6-P18⊂ ⊂L group, the PA signal in CD44v6-P18/AHC⊂ ⊂L group showed an obvious tumor boundary in five samples (Figure 6a and S15). The corresponding histopathology of biopsy from both side of the tumor boundary confirmed that the stronger PA signal was designated as tumor tissue and the other side was normal tissue (Figure S16). Furthermore, PA signal of CD44v6-P18/AHC⊂ ⊂L probe in tumor was ~18 folds enhancement compared with in normal tissue (T/N, ~18), which resulted in the significantly clear tumor boundary (Figure 6b). These data, all together, support the notion that CD44v6-P18/AHC⊂ ⊂L might be a promising supramolecular probe to accurately distinguish between tumor and normal tissue in BC. We concluded that, when the tumor was irradiation with MSOT laser, the energy of the laser was absorbed by P18 aggregates in the bilayer of liposomes and quickly converted into the heat, leading to the production of ultrasonic signal. Meanwhile, the heat induced the decomposition of AHC for sustained generation of CO2 bubbles in tumor, and the ultrasonic had the different backscatter between surrounding tissues and bubbles, thus resulting in the efficient amplification of PA signal and enhanced T/N ratio of tumor. Therefore, the sensitive PA probe realized the clear imaging of small tumor and tumor boundary, which provided the probability for complete tumor resection in clinic.


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Figure 6. a) The schematic illustration and the PA imaging of tumor boundary that treated with CD44v6 probe, (N = 5). Scale bars, 2 mm. b) PA signal in bladder normal and tumor tissue. The values were expressed as mean ± SD. Asterisks (*) denote the statistical significance: ***p < 0.001.

CONCLUSION In summary, we can clearly and effectively distinguish the small lesions (< 5 mm) and tumor boundary using PA imaging with signal self-amplifiable PA nanoprobe. The results demonstrated that using CD44v6-P18/AHC⊂ ⊂L as signal self-amplifiable PA nanoprobe, i) the PA signal in tumor sites is controllably enhanced due to bubble generation, ii) the imaging accuracy in bladder cancer lesions is promoted (< 5 mm), compared with conventional ultrasound and CT, and iii) importantly, the boundary of bladder tumor was clearly displayed by spatially controllable PA imaging with ~18 folds enhancement of T/N ratio. In this study, identification of human bladder cancer was highly performed through PA imaging with a high sensitive and specific probe which can be applied for guiding efficient surgery through precise tumor imaging, thereby significantly reducing the cancer recurrence. Therefore, based on the


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unique property of this probe, the tumor imaging of bladder cancer patients through instillation method is undergoing. ASSOCIATED CONTENT Supporting Information Experimental details, UV-Vis spectra, fluorescence spectra, heating/cooling curves, SEM images, PA imaging, cell viability assay, biodistribution of nanoprobes and results of histopathology evaluation. Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author [email protected], [email protected], [email protected] Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21674027, 21374026, 81771898 and 51573032), the National Science Fund for Distinguished Young Scholars (51725302), the Science Foundation of Heilongjiang Province (201624), the Key


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High-Performance Identification of Human Bladder Cancer Using

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