Photostable Iridium(III)-Cyanine Complex Nanoparticles for

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Biological and Medical Applications of Materials and Interfaces

Photostable Iridium(III)-Cyanine Complex Nanoparticles for Photoacoustic Imaging Guided Near-Infrared Photodynamic Therapy in Vivo Qi Yang, Hongyu Jin, Yucong Gao, Jiaomin Lin, Hong Yang, and Shi-Ping Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04098 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Applied Materials & Interfaces

Photostable Iridium(III)-Cyanine Complex Nanoparticles for Photoacoustic Imaging Guided Near-Infrared Photodynamic Therapy in Vivo Qi Yang, Hongyu Jin, Yucong Gao, Jiaomin Lin*, Hong Yang*, Shiping Yang* The Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai, 200234, China. ABSTRACT: The iridium (III)-cyanine complex (IrCy) was fabricated by conjugating an iridium (III) complex to a cyanine dye with an intense near-infrared (NIR) absorption. IrCy complex nanoparticles (NPs) with high water solubility and photo-stability were prepared by a solvent evaporation-induced self-assembly strategy. Considering their effective photacoustic (PA) imaging and generation of 1O2 property with 808 nm laser irradiation in aqueous solution, PA imaging guided NIR-driven photodynamic therapy in vivo was effectively conducted in the 4T1 xenograft model. We developed a real-time PA imaging methodology to investigate the pharmacokinetics, tumor targeting, and biodistribution of IrCy NPs. Taking advantage of the analysis of PA signal of the common iliac vein, the blood circulation halflife of IrCy NPs in mice was calculated to be ~18 h, and the enhanced permeability and retention effect of IrCy NPs offered the maximum targeting property in tumor at about 24 h. The obvious change of PA imaging signal in kidney and bladder confirmed IrCy NPs should be excreted partially from the urine system, and the PA signal decreased from 12.5 to 2.8 times in liver, and from 28.8 to 9.4 times in spleen also confirmed the hepatic metabolic pathway. KEYWORDS: Iridium-cyanine complex; near-infrared photodynamic therapy; nanoparticles; photoacoustic imaging; in vivo

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INTRODUCTION Photoacoustic (PA) imaging has received a great attention in the field of biomedical applications due to the combination advantages of the high spatial resolution of ultrasound and high sensitivity of optical imaging.1-5 These advantages are depended on the PA effect of endogenous or exogenous contrast agents. Although some endogenous agents (e.g. hemoglobin and melanin) can generate strong PA contrasts, there is not sufficient to provide more useful information especially in the detection of early stage tumors.6, 7 Therefore, the exploration of exogenous contrast agents is essential for PA imaging in vivo. Generally, exogenous PA contrast agents (CAs) can be categorized into small molecule organic dyes,8, 9 polymer-based nanomaterials,10-13 and metallic/semimetallic nanomaterials.14-20 Among them, small molecule organic dye is one of the most widely applied CAs. Notably, cyanine dyes are one of the most popular small molecule PA probes own to their intense absorption in the NIR region, and excellent biocompatibility and biodegradability.21,

22

However, cyanine dyes

suffer from the poor photo-stability. To address this issue, it is a promising strategy to construct cyanine dyes-based nanoparticles such as liposome,23 micelles,24 and etc. to enhanced the photostability. On the other hand, for photodynamic therapy (PDT), iridium complexes were generally preferred because of the heavy atom effect of iridium ions.25-28 Unfortunately, the conventional iridium complexes usually exhibited absorption band located in the UV or visible light region.27, 28 The limitation of tissue penetration depth and tissue harmness still obstructed the application of iridium complexes in vivo PDT. Therefore, the development of iridium complexes for NIR-PDT is an urgent task for in vivo cancer theranostics.29, 30 According to the above points, herein, we incorporated an iridium center into a cyanine dye to obtain iridium (III)-cyanine complex (IrCy) with the intense NIR absorption (Scheme 1). Under a solvent evaporation-induced self-assembly strategy, IrCy complex nanoparticles 2 ACS Paragon Plus Environment

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(IrCy NPs) with the good water solubility and photo-stability were prepared. Due to the intense NIR absorption, they exhibited strong PA signal that can be utilized for real-time investigating the pharmacokinetics, tumor targeting, and biodistribution of IrCy NPs, and effective generation of singlet oxygen (1O2) for PDT of tumor upon 808 nm laser irradiation (50 mW/cm2). This work establishes an alternative way for exploring the iridium complexes for in vivo cancer theranostics. RESULTS AND DISCUSSION Synthesis and Characterization of IrCy NPs. IrCy complex was synthesized by reaction of Cy-phen and [(ppy)2Ir(μ-Cl)2Ir(ppy)2] in dry CH3OH and CH2Cl2 mixed solution (Scheme 2), and characterized by 1H NMR, MS, and UV-Vis spectra (Figure S1). IrCy NPs were fabricated via a solvent evaporation-induced self-assembly strategy. During the evaporation of dichloromethane, IrCy was encapsulated in the hydrophobic PPO blocks of F127 to assembly into nanoparticles. The hydrophilic PEO units of F127 on the surface ensured the high water solubility of IrCy NPs. As presented in Figure 1a and 1b, the average diameter of the spherical IrCy NPs was about 200 nm. The hydrodynamic diameter in water was 216 ± 6 nm with a low PDI value (PDI = 0.18, Figure S2) evidencing a good homogeneity of IrCy NPs. Their zeta potential was 38.5 ± 0.2 mV (Figure S2), which proved the existence of the cationic IrCy complex in the nanoparticles. No obvious changes of the hydrodynamic diameter in PBS and RPMI 1640 medium plus 10% serum were observed within one week (Figure S3), respectively, which provided possibilities for in vivo application. Compared to the absorption spectrum of IrCy, similar weak visible absorption band centered at ~ 400 nm of IrCy NPs (Figure 1c) resulted from the triplet metal-to-ligand charge transfer (3MLCT) was observed. While the NIR absorption peak of IrCy complex was mainly located 820 nm, IrCy NPs had two bands located at 755 and 815 nm, respectively, due to partially H-type aggregations of IrCy in the nanoparticles. The absorbance intensity at 815 nm of IrCy NPs 3 ACS Paragon Plus Environment


remained stable within the pH range of 5.1-9.0 (Figure S4). Meanwhile, it decreased by only about 20% when irradiated by laser (λ = 808 nm, and power = 50 mW/cm2) for 30 min. However, a significant decrease was observed for the absorbance intensity of IrCy and Cy7.5 (a typical commercial NIR dye) decreased by ~50%, and ~96% (Figure 1d) treated by the same condition, respectively. The intense NIR absorption and enhanced photo-stability of IrCy NPs strongly promoted us to investigate the long term PA imaging and theranostics. As shown in Figure 1e, IrCy NPs in aqueous solution exhibited a strong PA signal upon irradiated by a pulsed laser irradiation with excited wavelengths of 680-900 nm. The maximum peak centered at 815 nm was also observed, which was consistent with that in the absorption spectrum. The intensity of PA signal and the concentration of IrCy NPs (from 0 to 12.4 μM of IrCy, ICP quantitative based on Ir3+) exhibit a linearly dependent relationship (Figure 1f), which offered the possibility for the quantitative analysis by virtue of PA imaging signals. PA imaging for tumor in vivo. In a 4T1 xenograft model, IrCy NPs (20 mg IrCy/kg body weight) or saline was intravenous (i.v.) injected, and then the blood circulation time of the nanoparticles was analyzed from the real-time PA imaging of the common iliac vein (Figure 2a, S5). Using the real-time PA signals, the blood circulation half-life time of IrCy NPs was estimated to be ~18 h (Figure 2b). Such a long circulation half-life is beneficial for the nanoparticles to be effectively passively targeted to tumor by EPR effect. In Figure 2c, in vivo PA images (under the 815 nm laser irradiation) of tumor were recorded before and after injection of IrCy NPs. The three-dimensional pictures showed that PA signals did not come from a single point or slice, but from the whole tumor. The areas marked by the red circle in these pictures were the tumor area in different time, in which a gradual increased PA signal with the time for the tumor area could be observed after i.v. injection. This indicated that IrCy NPs could be passively targeted to tumor. From the transverse view of tumor (Figure 2c), the PA signals can hardly be observed before injection since the NIR absorption from the 4 ACS Paragon Plus Environment

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oxyhemoglobin and deoxyhemoglobin are very low.31,32 After the injection, the tumor region exhibited clear PA images, and the signal increased with the prolonged circulation time. The brightest PA signal appeared at 24 h post-injection, which suggested that the optimal time for in vivo therapy was 24 h after i.v. injection. The quantitative analysis of the time-dependent change of the PA signal was determined using the origin software. As presented in Figure 2d, only after 3 h post-injection, the relative PA intensity for the tumor sites achieved nearly 3.2 times enhancement (the ratio of PA signal in tumor to that before injection), and reached the maximum value at 24 h. The high PA intensity indicated the much high uptake of IrCy NPs in tumor, which can be mainly attributed to the EPR effect.33-35 After 24 h post-injection, the relative PA intensity decreased gradually. Even at 108 h i.v., the relative PA intensity was still about 2.8 times. As shown from the PA signal distribution of Hb, HbO2 and IrCy NPs in tumor (Figure 2e), it was clear that IrCy NPs was located in the same region of HbO2, which proved that IrCy NPs should be mainly targeted in the oxygen enrichment area of the tumor. These facts provided a better window and environment to treat tumor using PDT. To study the clearance pathway of IrCy NPs in vivo, the bio-distribution in the 4T1 xenograft model was further quantitatively analyzed via the real-time PA imaging after i.v. injection (Figure 3a). For the kidney the relative PA intensity reached the maximum value of about 4.1 times at 30 h i.v. injection, and after that slowly decreased to about 2.0 times at 48 h i.v injection (Figure 3b and 3c). Simultaneously, for the bladder, the relative PA intensity at the time points of 84 h was about 12.6 times than that before injection (Figure 3d, e). The change of PA signal in kidney and bladder strongly suggested IrCy NPs should be excreted partially from the urine system.36 From 3 to 24 h i.v., the relative PA intensity in the liver increased from 4.3 to 12.5 times (Figure 3f and 3g), and increased from 4.6 to 28.8 times in the spleen, respectively (Figure 3h and 3i). After 150 h i.v., the relative PA intensity of liver and spleen decreased to 2.8 and 9.4 times, respectively. These bio-distributions of IrCy NPs in liver and 5 ACS Paragon Plus Environment


spleen suggested the partial hepatic metabolic pathway.37-39 The efficient renal and hepatic clearance of IrCy NPs was perhaps attributed to their small hydrodynamic diameter and highdensity PEG units decorated on the particle surface . PA Guided PDT in Vivo. The 1O2 generation ability of IrCy NPs in an aqueous solution was evaluated using a 1O2 indicator of ABDA. As shown in Figure 4a, 4b and S6, when irradiated via laser (λ = 808 nm, and power = 50 mW/cm2) for 15 min, the fluorescence intensity of ABDA in aqueous solution containing IrCy NPs (10 M) and ABDA (1 mg/mL) was decreased about ~95%, indicating an effective generation 1O2 of IrCy NPs. The generation of 1

O2 was further confirmed through the inhibition experiment (Figure 4b), which revealed that

the change of the fluorescence intensity of ABDA was very limited under the same condition upon the addition of a typical 1O2 quencher (NaN3, 32 M).40 Using indocyanine green as reference (1O2 generation efficiency, ΦΔ = 0.2%) and singlet oxygen sensor green (SOSG) as 1

O2 indicator, the ΦΔ of IrCy NPs was determined to be about 4.36% (Figure S7), which is

comparable with some of semiconducting polymer nanoparticles.41-44 Considering the excellent ability of the generation 1O2 of IrCy NPs in an aqueous solution, the effect of photodynamic therapy (PDT) for cancer cells was determined. Firstly, the cytotoxicity was evaluated using 4T1 and HUVEC cell lines with standard MTT assay, which revealed that there is no significant cytotoxicity was observed for these two cell lines (Figure S8). Similarly, IrCy NPs exhibited the excellent hemo-compatibility within our experimental concentration range (Figure S9). Secondly the generation of 1O2 in cancer cells was evaluated. 4T1 cells were incubated with SOSG (1O2 indicator, 20 μM) and IrCy NPs (0, 5, 10, 25, and 50 μM of IrCy, respectively). According to Figure 4c, upon the laser irradiation (λ = 808 nm, and power = 50 mW/cm2), the fluorescence intensity of 4T1 cells increased from 2.5 to 87.8% with the increase of the incubation concentration from 2 to 50 μM. Thirdly, the laser ablation of 4T1 cells was evaluated by trypan blue staining. As expected, the cell viability for the group IrCy 6 ACS Paragon Plus Environment

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NPs (25 μM) treated only and the group of laser irradiation only displayed no significant difference (Figure 4e). However, most of the cells were stained for the group with IrCy NPs incubation (25 μM, 6 h) and then laser irradiation for 20 min. To confirm the effect of the PDT, the cell viability for the group of IrCy NPs treatment only and the group of IrCy NPs treatment and then laser irradiation was quantitatively determined via MTT assay. As presented in Figure 4d, the cell viability for the group of IrCy NPs treatment only remained above 80%, while for the group of IrCy NPs treatment and then laser irradiation decrease from 65.5 to 26.7% with the incubation concentration of IrCy NPs increasing from 5 to 50 μM. All these data suggested that IrCy NPs were ideal choice for the application in PDT. On the basis of the property of PA imaging and the effective generation of 1O2 , in vivo PDT was possibly carried out using a 4T1 xenograft model. To evaluate the efficiency of PDT, the time-dependent change of the tumor volume was monitored after PDT. At the 15th day, the relative tumor volumes exhibited an increase of ~15, ~11, and ~10 times in the PBS group, IrCy NPs group and PBS + laser group, respectively, as compared with those at the first day (Figure 5a). In strong contrast, the relative tumor volume for the PDT group was noticeably restrained to ~2 times, as compared with that before PDT. After PDT, the mice for all groups were sacrificed for further evaluating the effect of PDT. The weight of the tumors for PDT group was 13 times smaller than that in PBS group, indicating that the tumor growth for this group was suppressed greatly (Figure 5b). Under the same condition of PDT, the change of the tumor temperature is very small (Figure S10), excluding the possibility of the photothermal therapy effect for tumor. Moreover, both H&E stain of main organs (Figure 5f) and the weight change were observed after PDT for 14 days (Figure S11), indicating that the side effect during PDT is low. Finally, the tumor that extracted from the mice was subjected to TUNEL analysis to confirm the efficacy of PDT. As shown in Figure 5d, e, the TUNEL positive cells were found to less than 20% in the three control groups, while that of PDT 7 ACS Paragon Plus Environment


group is about 77.2±3.7%, indicating that the PDT for the cancer therapy in the mice mode is effective even within such a short time. CONCLUSION In summary, we successfully fabricated a stable and effective nano-theranostic agent of IrCy NPs with for PA imaging-guided NIR-PDT in vivo. In a 4T1 xenograft mice model, IrCy NPs showed long blood circulation half-time and good targeting ability to tumor. Proved by the real-time PA imaging, IrCy NPs were able to be excreted partially from the urine and hepatic system. Due to their effective generation of 1O2 and targeting effect, IrCy NPs displayed excellent tumor ablation ability in the mice mode. This work highlighted that the metal complex nanoparticles are promising agent for cancer theranostics. EXPERIMENTAL SECTION The 1O2 generation in an aqueous solution: The 1O2 generation of IrCy and IrCy NPs was monitored through detecting the change of fluorescence intensity of ABDA excited by 360 nm. The ABDA and IrCy/IrCy NPs were dissolved in H2O with concentration of 1 mg/mL and 10 μM, respectively. Then, 50 μL of ABDA and 4 mL of IrCy/IrCy NPs was mixed, and then irradiated by laser (λ = 808 nm, and power = 50 mW/cm2) for 15 min. Finally, the fluorescence intensity of ABDA was measured on a fluorescence spectrophotometer with excited wavelength of 360 nm. With the same experimental condition, an IrCy NPs solution that contained ABDA and NaN3 (32 μM) was used as the control experiment. The 1O2 generate quantitative analysis of IrCy NPs was performed with commercial cyanine indocyanine green (ICG) as a contrast. The fluorescence probe of singlet oxygen sensor green (SOSG) was used as the 1O2 indicator. IrCy NPs and ICG were dissolved in distilled water with concentrations of 10 μg/mL, respectively. SOSG (5 μL, 100 μM) was added to IrCy NPs (1 mL) or ICG (1 mL) solution, following by irradiation by 808 nm laser 8 ACS Paragon Plus Environment

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(50 mW/cm2). The fluorescence intensity at 520 nm was recorded after every 10 seconds laser exposure. The enhancement of fluorescence intensity of SOSG was measured to calculate the 1

O2 generation efficiency (ΦΔ) using the following equation: ΦΔSPN-Ms = (rIrCy

NPs

NPs⁄AIrCy

)/(rICG⁄AICG )*ΦΔICG. The ΦΔICG was the 1O2 generation efficiency of ICG, which was

known as 0.002 according to previous studies.43 The rIrCy NPs and rICG were the reaction rate of SOSG with 1O2 generated from IrCy NPs and ICG, respectively. The AIrCy NPs and AICG were defined as corresponding absorbance of IrCy NPs and ICG at 808 nm, respectively. MTT assay: Two cell lines of 4T1 and HUVEC were used to investigate the in vitro cytotoxicity through standard MTT assay. Firstly, 4T1 or HUVEC cells that seeded in plates (96-well, with per well of about 1×104 cells) were cultured in the medium of DMEM for 24 h at 37 oC. After removing the medium, another 100 μL fresh medium that contained IrCy NPs with concentration of 0, 2, 5, 10, 25, and 50 μM, respectively, was added. The cells were further incubated in the medium for another 12 and 24 h, respectively. After that, the supernatant in the wells was removed slowly, and then 20 μL of MTT (with concentration of 5 mg mL-1) was added. Subsequently, the cells were cultured in the medium for another 4 h. After removing the supernatant, each wall was added with 100 μL of DMSO for dissolving the formazan. The characteristic absorption peaks at 492 nm for formazan was used to the measure the amount of the forming formazan. For evaluation of the phototoxicity of IrCy NPs, the 4T1 cells that cultured in 96-well plate were then incubated for 6 h in the serum-free medium contained IrCy NPs (25 μM). After removing the supernatant, fresh RPMI 1640 (100 μL) was putted into each well. Subsequently, the cells in the well were laser irradiated (λ = 808 nm, and power = 50 mW/cm2) for 10 min, and then cultured for additional 1 h. Finally, the cells were collected and their viability was determined through standard MTT assay. Hemolysis Assay: All procedures for the treatment of animal were carried out in accordance with the international standard. Fresh blood with volume of about 1 mL was obtained from 9 ACS Paragon Plus Environment


mice through extracting the eyeball blood. The obtained blood was then centrifuged to obtain the red blood cells (RBCs). After washed by PBS four times, the isolated RBCs were kept in 2 mL of fresh PBS. Then, in four centrifuge tube contained 0.8 mL PBS solution, 100 μL of IrCy NPs (5, 10, 25, and 50 μM) in PBS and 100 μL of RBCs suspension were added and shocked at 37 oC for 2 h. After that, the suspensions in centrifuge tube were centrifuged for 15 min with a rotate speed of 1500 rpm. Subsequently, the amount of released hemoglobin were determined through analyzed the supernatant (100 μL) of each centrifuge tube using a microplate reader. The positive and negative controls were used water and PBS, respectively. The percentage hemolysis was calculated by: Hemolysis (%) = (OD576sample -OD576negative control)/(OD576positive control-OD576negative control)×100%.

Flow cytometry: The efficiency uptake of IrCy NPs for the 4T1 cells was determined through flow cytometry analysis. In a typical experiment, 4T1 cells that prior seeded in 6-well plates were cultured in medium without serum but contained different concentrations (0, 2, 5, 10, 25, and 50 μM, respectively) of IrCy NPs. After 6 h, the medium in the well was removed, and fresh PBS was added into the wall. After washing by PBS, the 4T1 cells in the wall were harvested and re-suspended in PBS, and then analyzed using flow cytometry (Beckman). Trypan blue staining: The photodynamic therapeutic effect of cell level was confirmed by trypan blue staining. Similar with the previous procedure, in a 12 well plate, 4T1 cells were seeded and then incubated with IrCy NPs. After removed the medium, fresh PBS was added. For PDT, the cells in the walls were laser irradiated (λ = 808 nm, and power = 50 mW/cm2) for 20 min. Subsequently, the cells in the well were cultured for additional one hour and then stained by added 1 mL trypan blue solution (0.4%). The medium were removed after 10 min. Finally, the stained cells were imaged and counted for evaluating the efficiency of PDT. PA imaging: The PA imaging was carried out using a Multispectral Optoacoustic Tomography scanner (MSOT Invision 128, iThera medical, Germany). For in vitro imaging, IrCy NPs that dispersed in aqueous with the concentration of 0, 1.39, 3.94, 6.52, 7.72, 9.27, 10 ACS Paragon Plus Environment

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9.94, and 12.43 μM, respectively, was mixed with 3% agarose and transferred to a plastic pipe for phantom. The wavelength of the pulsed laser was 815 nm. A linear model-based inversion method was used to reconstruct the images. For in vivo imaging, the mice that bear 4T1 tumor were first anaesthetized with 5% isoflurane, and then imaged to obtain the best imaging section and the blank imaging phantoms. After intravenous injection of IrCy NPs (200 μL, 1.37 mM of IrCy), the PA imaging of the mice were collected at different times. The obtained images were analyzed on MSOT Invision 128 system. The parameters for the images were followed: surface fluence, 20 mJ cm-2; pulse energy, 70–120 mJ; central frequency, 5 MHz. Photodynamic therapy: The photodynamic therapy for the 4T1 tumor-bearing mice was carried out by four groups for comparing the treatment results. The laser group refers to group of mice irradiated by laser only. The PBS group is the group of mice injected with PBS (200 μL) via the tail vein only. The IrCy NPs and IrCy NPs+Laser groups refer to the group of mice injected with IrCy NPs (200 μL, 1.37 mM) only, and group of mice injected with IrCy NPs (200 μL, 1.37 mM) and then laser irradiation, respectively. For the groups contained laser irradiation, the irradiation were carried out at the tumor sites for 30 min using laser (λ = 808 nm, and power = 50 mW/cm2) after 24 h of injection. After 48 h, the mice for the two material groups were intravenously injected with 200 μL IrCy NPs (1.37 mM of IrCy) again. After 72 h, the tumor of the mice was irradiated for 30 min again. To evaluate the therapeutic effect, the mice weight and tumor volume (Volume = length×width2/2) for all groups were recorded every two days. After therapy, all of the mice for the four groups were sacrificed, and their viscera and tumor were excised for further TUNEL and H&E staining.

ASSOCIATED CONTENT Supporting Information. Experimental procedures for the synthesis and characterization of IrCy complex and IrCy NPs; absorption spectra of Cy-phen and IrCy; zeta potential, 11 ACS Paragon Plus Environment


hydrodynamic diameter and stability characterization of IrCy NPs; PA imaging of mice intravenous injected with saline; fluorescence spectra of ABDA with the presence of IrCy NPs and NaN3; fluorescence intensity of SOSG with the presence of ICG or IrCy; 4T1 and HUVEC cell viability; hemolysis of IrCy NPs; the body weight of mice and weight of main organs on the last day after PDT. These data are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was partially supported by National Natural Science Foundation of China (Nos. 21877080 and 21671135), Shanghai Sailing Program (17YF1413700), Shanghai Engineering Research Center of Green Energy Chemical Engineering, the Ministry of Education of China (IRT_16R49), Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, and International Joint Laboratory on Resource Chemistry (IJLRC). REFERENCES [1] Jiang, Y.; Pu, K. Multimodal Biophotonics of Semiconducting Polymer Nanoparticles. Acc. Chem. Res. 2018, 51, 1840-1849.


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Scheme 1. Schematic illustration of (a) the fabrication of IrCy NPs and (b) its application for PA-guided photodynamic therapy in vivo.


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Scheme 2. Synthesis of IrCy.



Figure 1. SEM (a) and TEM imaging (negative staining with 1% phosphotungstic acid) (b) of IrCy NPs. (c) The absorption spectra of IrCy (DMSO:H2O=1:49, v/v), and IrCy NPs in H2O. (d) The photo-stability of IrCy, IrCy NPs, and Cy7.5 after laser irradiation for 30 min (808 nm, 50 mw/cm2). (e) The PA spectrum of IrCy NPs with the concentration of 10 μM. (f) Concentration of IrCy NPs dependent PA signal intensity upon 815 nm plused laser irradiation. Inset: PA imaging of different concentrations of IrCy NPs embedded in agar gel cylinders.


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Figure 2. (a) Schematic views of the tumor bearing mouse during PA imaging. (b) Quantitative analysis of PA signals in the common iliac vein of the mouse. (C) PA imaging of tumor from different section after intravenous injection of IrCy NPs at 0, 3, 9, 24, 84, and 132 h, respectively. (d) The quantitative changes of the relative PA signals in tumor correspond to (c). (e) The PA imaging of Hb, HbO2, and IrCy NPs in tumor. The red dotted area was tumor.



Figure 3. (a) The transverse (xy) and longitudinal view (yz) of the mouse for quantitative analysis of different organs were acquired from 37 to 75 mm. The letters of H, L, K, B, S refer to heart, liver, kidney, bladder, and spleen, respectively. The time-dependent change of the relative PA imaging signals in kidney (b), bladder (d) liver (f), and spleen (h) after injection of IrCy NPs (20 mg/kg IrCy) via the tail vein. The signal of IrCy NPs was quantified


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as a function of time in the ROIs drawn around the (c) kidney (pink line), (e) bladder (brown line), (g) liver (yellow line) and (i) spleen (blue line).

Figure 4. (a) The time-dependent fluorescence spectra of ABDA(16 M) irradiated by laser in the presence of IrCy NPs (10 M). (b) The time-dependent singlet oxygen generation of IrCy NPs (10 M) without (red) and with (black) NaN3 (12.9 M,) after laser irradiation. (c) The mean phosphorescence intensity of 4T1 cells after incubation with various concentrations of IrCy NPs for 4 h. (d) The cell viability for 4T1 cells that cultured with different concentrations of IrCy NPs (0, 5, 10, 25, and 50 μM, respectively) and then laser irradiation. (e) The optical images of 4T1 cells irradiated by laser for 20 min (e1), 4T1 cells stained by 23 ACS Paragon Plus Environment


trypan blue after incubation with 25 M IrCy NPs (e2), and 4T1 cells incubated with 25 M IrCy NPs irradiated by laser for 20 min (e3). Scale bar: 100 μm.

Figure 5. (a) Time-dependent change of tumor volume after different treatment. (b) The weight of tumor on the last day after PDT for four groups. (c) Mice photographs of four groups during PDT. TUNEL stain images (d) and quantitative analysis (e) of tumor slices. PBS group: mice injected with 200 μL PBS only via the tail vein; IrCy group: mice injected with IrCy NPs (20 mg/kg of body weight) only; Laser group: mice irradiated by laser only; 24 ACS Paragon Plus Environment

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IrCy NPs+Laser group; mice injected with IrCy NPs and then irradiated by laser (f) H&E staining of the main organs excised from the mice for the four groups on the last day after PDT. Scale bar: 100 μm.



Table of Contents Graphic


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The iridium (III)-cyanine complex (IrCy) nanoparticles (NPs) were fabricated for photoacoustic imaging guided near-IR photodynamic therapy in Vivo.

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Photostable Iridium(III)-Cyanine Complex Nanoparticles for

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