NIR-Light-Triggered Anticancer Strategy for Dual-Modality Imaging

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NIR light-triggered anticancer strategy for dual-modality imagingguided combination therapy via a bioinspired hybrid PLGA nanoplatform Xue Shen, Tingting Li, Zhongyuan Chen, Xiaoxue Xie, Hanxi Zhang, Yi Feng, Shun Li, Xiang Qin, Hong Yang, Chunhui Wu, Chuan Zheng, Jie Zhu, Fengming You, and Yiyao Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01321 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

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Molecular Pharmaceutics

NIR light-triggered anticancer strategy for dual-modality imaging-guided combination therapy via a bioinspired hybrid PLGA nanoplatform

Xue Shen1, Tingting Li1, Zhongyuan Chen1, Xiaoxue Xie1, Hanxi Zhang1, Yi Feng1, Shun Li1,3, Xiang Qin1,3, Hong Yang1,3, Chunhui Wu1,3, Chuan Zheng2, Jie Zhu2 Fengming You2, Yiyao Liu1,2 *

1School

of Life Science and Technology, University of Electronic Science and

Technology of China, Chengdu 610054, Sichuan, P. R. China; 2Hospital of Chengdu University of Traditional Chinese Medicine, No. 39 Shi-er-qiao Road, Chengdu 610072, Sichuan, P. R. China; 3Center for Information in Biology, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, P. R. China

* To

whom correspondence should be addressed:

Prof. Yiyao Liu, Ph.D, School of Life Science and Technology, University of Electronic Science and Technology of China, and Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, P. R. China. Tel: +86-28-8320-3353, fax: +86-28-83208238, E-mail: [email protected] or [email protected]

1

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A promising approach towards cancer therapy is expected to integrate imaging and therapeutic agents into a versatile nanocarrier for achieving improved antitumor efficacy and reducing the side effects of conventional chemotherapy. Herein, we designed a poly(D,L-lactic-co-glycolic acid) (PLGA)-based theranostic nanoplatform using the double emulsion solvent evaporation method (W/O/W), which is associated with bovine serum albumin (BSA) modifications, to co-deliver indocyanine green (ICG), a widely used near-infrared (NIR) dye, and doxorubicin (Dox), a chemotherapeutic drug, for dual-modality imaging-guided chemo-photothermal combination cancer therapy. The resultant ICG/Dox co-loaded hybrid PLGA nanoparticles (denoted as IDPNs) had a diameter of around 200 nm and exhibited excellent monodispersity, fluorescence/size stability, and biocompatibility. It was confirmed that IDPNs displayed a photothermal effect and that the heat induced faster release of Dox, which led to enhanced drug accumulation in cells and was followed by their efficient escape from the lysosomes into the cytoplasm and drug diffusion into the nucleus, resulting in a chemo-photothermal combinatorial therapeutic effect in vitro. Moreover, the IDPNs exhibited a high ability to accumulate in tumor tissue, owing to the enhanced permeability and retention (EPR) effect and could realize real-time fluorescence/photoacoustic imaging of solid tumors with a high spatial resolution. In addition, the exposure of tumor regions to NIR irradiation could enhance the tumor penetration ability of IDPNs, almost eradicating subcutaneous tumors. In addition, the inhibition rate of IDPNs used in combination with laser irradiation against EMT-6 tumors in tumor-bearing nude mice (chemo-photothermal therapy) was approximately 95.6%, which was much higher than that for chemo- or photothermal treatment alone. Our study validated the fact that the use of well-defined IDPNs with NIR laser treatment could be a promising strategy for the early diagnosis and passive tumor-targeted chemophotothermal therapy for cancer.

Keywords: PLGA, on-demand drug release, combination therapy, dual-modality imaging, tumor accumulation, penetration 2


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1. Introduction Cancer theranostics, the combinatorial study of diagnostics and therapeutics, represents an emerging medical need in recent years.1, 2 Thus, many theranostic agents, including near-infrared (NIR) light-absorbing agents, iron nanoparticles, and radionuclides have been developed and widely used for cancer diagnosis and therapy. 3, 4

Notably, most NIR light-absorbing agents are capable of converting light energy

into thermal energy and/or generating reactive oxygen species (ROS) after exposure to NIR laser irradiation,5-7 realizing photothermal therapy (PTT) and/or photodynamic therapy (PDT) for directly killing cancer cells by focusing the NIR laser beam on tumor regions. The use of the NIR light implies that the light has a wavelength range of 650950 nm, at which the skin and tissue show minimal light absorption, resulting in low phototoxicity in skin and tissue.4 Meanwhile, the light in the NIR window has multiple advantages, including high spatiotemporal precision, real-time dosage adjustment, and deep tissue penetration.8, 9 Additionally, NIR light-induced photothermal therapy (PTT), as a minimally invasive treatment, can utilize the heat generated by the laser irradiation of NIR light-absorbing agents to kill cancer cells directly.10, 11 Moreover, the production of local hyperthermia can not only enhance the sensitivity of cancer cells to chemotherapeutics, but also increase the penetration of drugs into tumors, by affecting the permeability of the tumor vascular and cell membrane.12 In addition, the ROS generated because of exposure to NIR irradiation from the photosensitizer, which is used widely as a light-heat converting material in photothermal therapy, are capable of destroying the endosomal/lysosomal membrane and facilitating drug escape from 3



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endosomes/lysosomes.13 Until now, a variety of NIR light-absorbing agents, ranging from inorganic nanoagents (i.e., gold nanomaterials14, carbon nanomaterials15, and CuS nanomaterials16) to organic molecules (i.e., perylene diimide (PDI)17 and indocyanine green (ICG)18) have been investigated in various tumor models. Although these inorganic nano-agents have shown encouraging results in many animal studies, they have struggled to be of use in clinical settings. For example, gold nanomaterials potentially exhibit long-term toxicity, because they are not biodegradable and not effectively cleared out from the body, which hinders their clinical application.17, 19 Among these NIR light-absorbing agents, ICG, a NIR cyanine dye that has been approved by the U.S. Food and Drug Administration (FDA), is a perfect theranostic agent that has been developed for NIR fluorescence imaging-guided antitumor applications. The strong NIR absorbance of ICG endows them with multifunctional properties, including photothermal effects, and NIR fluorescence imaging and photoacoustic imaging properties.17,

20, 21

However, ICG

molecules are susceptible to self-bleaching and are unstable in aqueous solutions, and have other drawbacks, including poor photostability, rapid elimination from the body, and lack of targeting, which result in them having limited clinical applications.22-24 Therefore, numerous nanocarriers, including liposomes, micelles, and vesicles have been developed to cope with these issues, by effectively delivering ICG for both fluorescence imaging and photothermal therapy (PTT).25-27 Recently, the use of versatile nanocarriers as drug delivery systems integrating NIR light-absorbing agents and therapeutic agents for chemo/photothermal 4


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combination therapy has received considerable attention in the field of nanomedicine.28 Doxorubicin (Dox), a first-line, widely used chemotherapeutic agent approved by the FDA, is capable of inhibiting or killing malignant cells, by preventing DNA replication and cell division, through its DNA intercalation ability.29,

30

However, multidrug

resistance of tumor cells is one of the main reasons for the failure of chemotherapy31 and the severe side effects of Dox, including cardiotoxicity, myelosuppression, mucositis, and alopecia, because its non-specific drug distribution might lead to the development of limited clinical applications of Dox.32-34 Thus, the development of nanocarriers that co-delivered ICG and Dox has become an efficient strategy to enhance ICG stability, minimize the side effects of Dox, and increase their tumor accumulation through passive targeting. Because of passive targeting, nanoparticles can penetrate the interstitium and finally become entrapped in tumors, because their sizes are smaller than the fenestrations of vascular endothelial cells of tumors, where the leaky vasculature and poor lymphatic drainage contribute to the enhanced permeability and retention (EPR) effect.35 Although various nanocarriers have been fabricated to codeliver therapeutic drugs and NIR light-absorbing agents for chemo-photothermal therapy, several challenges need to be addressed, including their complicated synthesis process, non-biodegradability, and lack of selective drug release. For example, Yu Y et al36 reported a nano-sized system termed as Dox/ICG@biotin-PEG-AuNC-PCM, prepared by filling the interior of AuNCs with Dox, ICG, and 1-tetradecanol, and modifying the surface with biotinylated poly (ethylene glycol) via Au-S bonds, which requires a complicated multistep synthesis. Baek S et al37 designed a hybrid system by 5



coating the NIR responsive gold nanorods with drug reservoirs, i.e. mesoporous silica shells, to load Dox for chemo-photothermal therapy and diagnostic applications. However, inorganic nanomaterials (gold and silica) are not biodegradable and might raise long-term toxicity and safety concerns. Additionally, polymeric nanoparticles are extensively used to encapsulate drugs and imaging agents through the self-assembly process, but their uncontrolled drug release behavior might contribute to their compromised anticancer effect.38, 39 Herein, with the aim of meeting these challenges, a simple and multifunctional theranostic nanoplatform was fabricated, based on poly(D,L-lactic-co-glycolic acid) (PLGA), which is biocompatible and biodegradable.40 We described the water-in-oilin-water (W/O/W) double emulsion solvent evaporation method associated with bovine serum albumin (BSA) modifications to produce the bioinspired hybrid PLGA nanoparticles to co-load ICG, a widely used NIR dye, and Dox, a chemotherapy drug for both high contrast fluorescence/photoacoustic imaging and combined thermochemotherapy (Fig. 1). BSA modified on the surface of PLGA nanoparticles acted as a surface stabilizer and biocompatible cover and was able to bind the ICG molecules noncovalently. As depicted in Fig. 1, the BSA modified ICG/Dox co-loaded hybrid PLGA nanoparticles (denoted as IDPNs) could accumulate into tumor regions via the EPR effect after being intravenously injected into mice, and distribute into the endo/lysosomal compartments via the endocytosis mechanism. IDPNs, which were regulated by the external NIR laser irradiation, released more drugs, owing to the production of hyperthermia by the enhanced photothermal effect of ICG and generated 6


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singlet oxygen, leading to the disruption of lysosomes. Thus, more nanoparticles and released Dox escaped into the cytoplasm, and Dox then diffused to the cell nucleus, where it exhibited cytotoxic effects. The photothermal effect, drug release behavior, intracellular distribution, and cytotoxicity of IDPNs were investigated. The fluorescence/photoacoustic imaging capability, biodistribution, antitumor effect, and the intratumoral distribution of IDPNs were further evaluated in vivo. Our results corroborated the fact that the designed IDPNs showed great promise as an efficient theranostic nanoplatform for passive tumor-targeted and dual-modality imaging-guided chemo-photothermal combination therapy for cancer.

2. Materials and methods 2.1. Materials PLGA (50:50, inherent viscosity 0.20 dL/g, MW=15,000) was obtained from the Shandong Institute of Medical Instruments (Shandong, China), poly(vinyl alcohol) (PVA) (MW=30,000-70,000, 78%-90% hydrolyzed); indocyanine green (ICG) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Doxorubicin hydrochloride (Dox) was obtained from Hisun Pharmaceuticals (Zhejiang, China). Chlorpromazine and cytochalasin D were acquired from Enzo Biochem (New York, NY, USA). LysoTracker Green and Calcein AM were purchased from Life Technologies (Grand Island, NY, USA). Cell culture media such as Roswell Park Memorial Institute 1640 (RPMI 1640), Dulbecco's modified Eagle’s medium (DMEM) containing high glucose levels, fetal bovine serum (FBS), and trypsin were 7



obtained from Gibco (Grand Island, NY, USA). The 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and Annexin V-FITC/PI staining kits were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals and solvents were used as received without performing further purification processes, unless otherwise noted. The mouse embryonic fibroblast cells (NIH-3T3), human cervical carcinoma cells (HeLa), and murine mammary cancer cells (EMT-6) were obtained from the American Type Culture Collection (Manassas, VA, USA).

2.2. Preparation of IDPNs IDPNs were prepared by the water-in-oil-in-water (W/O/W) double emulsion method, according to our previously published procedures with slight modifications.1, 29, 41

Briefly, PLGA (100 mg) was directly dissolved in 2 mL methylene chloride, after

which 40 μL of Dox aqueous solution (20 mg/mL) and 150 μL of ICG aqueous solution (2 mg/mL) were added to the organic phase; subsequently, the mixture was emulsified by sonication with 35% amplitude for 2 min, using a Digital Sonifier S-250D (Branson Ultrasonic, Danbury, CT, USA) in an ice bath to obtain the primary emulsion (first emulsion). Next, this primary emulsion was immediately poured into 10 mL of PVA solution (3%, w/v) containing 1.2 mg ICG and 4 mg BSA and sonicated for 3 min to form a double emulsion (second emulsion). After magnetic stirring of the final emulsion was done overnight to fully evaporate the organic solvent, the BSA modified ICG/Dox co-loaded hybrid PLGA nanoparticles (denoted as IDPNs) were collected by performing centrifugation at 1200 rpm for 15 min and washed three times with deionized water. The same procedures were used to prepare the ICG loaded hybrid 8


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PLGA nanoparticles in the absence of Dox (denoted as IPNs) or the Dox loaded hybrid PLGA nanoparticles in the absence of ICG (denoted as DPNs). Hybrid PLGA nanoparticles without loaded ICG/Dox (denoted as PNs) and ICG/Dox co-loaded PLGA nanoparticles without BSA modifications (denoted as (ICG/Dox)/PLGA) were also prepared in the same manner. The supernatants of the emulsion obtained after performing centrifugation and the three washing steps were collected and the levels of non-entrapped Dox and ICG in the supernatants were determined by a UV-visible spectrometer (UV-2910; Hitachi, Tokyo, Japan) at 488 or 780 nm. The encapsulation efficiency (EE) of Dox or ICG (percentage of the loaded Dox or ICG out of that used to prepare IDPNs) was calculated using the following formula: EE = (weight of the drug in the nanoparticles/initial feeding amount of drug) × 100%

2.3. Characterization The morphology and size of the as-prepared nanoparticles were characterized using both scanning electron microscopy (SEM) (Helios NanoLab 650; FEI, Eindhoven, Netherlands) and transmission electron microscopy (TEM) (JEM-100CX; JEOL, Tokyo, Japan). The size and zeta-potential distribution were examined using a ZetaPlus particle size and zeta potential analyzer (Brookhaven Instruments, Holtsville, NY, USA), in accordance with the instructions in the manufacturer’s operating manual. The absorption spectra of free Dox, free ICG, PNs, IPNs, and IDPNs were obtained using a UV-visible spectrometer (UV-2910; Hitachi, Tokyo, Japan). In vitro photoacoustic imaging and photoacoustic signal intensity of IDPNs with different ICG concentrations were obtained using a multispectral optical tomography system (MSOT inVision 128, 9



iThera Medical, Germany).

2.4. In vitro photothermal effect The photothermal effects of free ICG, IPNs, DPNs, and IDPNs were evaluated by measuring the temperatures of each solution (200 μL) at an ICG concentration of 15 μg/mL, under 808 nm laser irradiation (1.5 W/cm2). Water was used as the control in this experiment. The temperature was measured using a digital thermometer (HH906AU, Omega) at different time points. The infrared thermographic maps of water, free ICG, IPNs, and IDPNs stored in a 96-well plate at an ICG concentration of 15 μg/mL under laser irradiation (808 nm, 1.5 W/cm2) for 10 min were recorded using an infrared thermal imaging camera (Ti27, Fluke, USA). Meanwhile, the photothermal effect of IDPNs at different ICG concentrations (10, 15, 20 μg/mL) under 808 nm laser irradiation (1.5 W/cm2) or of IDPNs under 808 nm laser irradiation at different power intensities (0.5, 1, 1.5, 2 W/cm2) were also evaluated, by measuring the temperatures using a digital thermometer (HH906AU, Omega) at different time points.

2.5. NIR light-triggered drug release In vitro Dox release profiles of IDPNs were determined by immersing the IDPNs in test tubes that contained 2 mL of buffer solution (PBS solution at pH 7.4 or acetate buffer solution at pH 4.6). The test tubes were gently shaken in a thermostatic rotary shaker at 100 rpm and 37 ºC. At the prescribed time points, the samples were irradiated using an 808 nm laser for 5 min at a power density of 1.5 W/cm2. The supernatants in 10


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each test tube were collected by centrifugation and replaced with fresh buffer solution at different time intervals. The amount of Dox released in the supernatants was measured using a UV-visible spectrometer (UV-2910; Hitachi, Tokyo, Japan) at 480 nm and the accumulative percentage of the released Dox was presented as a function of the incubation time. Meanwhile, the Dox release profiles from IDPNs (with BSA modification) or (ICG/Dox)/PLGA nanoparticles (ICG/Dox co-loaded PLGA nanoparticles without BSA modification) under pH 7.4 that were not exposed to laser irradiation at predetermined time points were also determined by a method that was similar to that described above.

2.6. Cellular experiments To observe the cellular internalization of IDPNs in EMT-6 cells, EMT-6 cells (8×105 cells/well) were seeded in a glass-based 24-well plate and incubated at 37 ºC in the presence of 5% CO2 overnight, to allow cell attachment. Then, the medium was replaced with fresh medium containing free ICG (15 μg/mL), free Dox (3.9 μg/mL), and IDPNs with the same concentration of Dox and ICG and incubated at 37 ºC for 4 h. Another group of IDPN treated cells was exposed to irradiation for 3 min (808 nm, 1.5 W/cm2) after an incubation period of 2 h. After a total incubation period of 4 h, all the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde for 20 min. Then, the cells were washed and observed using a confocal laser scanning microscope (CLSM; Leica SP5II, Wetzlar, Germany). 11



For the intracellular distribution assay of IDPNs, EMT-6 cells seeded overnight in a glass-based 24-well plate were treated with fresh medium containing IDPNs (15 µg/mL of ICG, 3.9 µg/mL of Dox) and incubated at 37 ºC for 1 h, and were subjected to the presence or absence of irradiation for 3 min (808 nm, 1.5 W/cm2). Then, the cells were washed and further incubated with 100 nM of LysoTracker Green for 20 min to stain lysosomes; the excitation wavelength of LysoTracker Green was 488 nm. The fluorescent images of cells were acquired by CLSM. To investigate the cellular internalization mechanisms of the IDPNs, the cellular uptake assay was performed according to our previously reported literature.42 EMT-6 cells (3×104 cells/well) were seeded in 48-well plates and incubated for 24 h. Then, the cells were treated with the different endocytosis inhibitors, including 10 µg/mL chlorpromazine for the inhibition of clathrin-mediated endocytosis, 50 µg/mL genistein or 15 µg/mL nystatin for the inhibition of caveolae-mediated endocytosis, 10 µg/mL cytochalasin D for the inhibition of macropinocytosis, and 0.1% sodium azide (NaN3) in serum-free RPMI 1640 medium for 30 min at 37 ºC, or treated without any endocytosis inhibitors in serum-free RPMI 1640 medium at 4 ºC. NaN3 (energy inhibitor) or treatments at 4 ºC were employed to block cellular adenosine triphosphate (ATP) synthesis. Cells treated without any cellular uptake inhibitors at 37 ºC were used as controls. Cells were then rinsed with PBS and further incubated with IDPNs (15 µg/mL of ICG, 3.9 µg/mL of Dox) in fresh medium for 4 h. Finally, the fluorescent images of cells were examined using an inverted fluorescent microscope (Nikon TE12


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2000U). For quantitative analysis, cells were lysed with 0.5% (w/v) sodium dodecyl sulfate (pH 8.0), and the intensity of cellular fluorescence was subsequently detected using a fluorescence spectrophotometer (Hitachi F-7000).

2.7. Cytotoxicity assay The cell viability of EMT-6 cells after the administration of different treatments was determined using a cell counting kit-8 (CCK-8) (Beyotime, Jiangsu, China), in accordance with the manufacturer’s instructions. Specifically, EMT-6 cells were plated in a 96-well plate at a density of 1×104 cells per well and incubated at 37 ºC in the presence of 5% CO2 for 24 h, to allow them to adhere to the wells. After removing the culture medium, EMT-6 cells were treated with 200 µL of fresh medium containing free ICG, IPNs, and IDPNs at ICG concentrations of 5, 10, and 15 µg/mL (the corresponding Dox concentration was 1.3, 2.6, and 3.9 µg/mL) for 12 h. The cells that were not subjected to any treatment were used as controls. Afterward, the medium was removed, and the cells were washed with PBS and replenished with fresh medium. Then, free ICG, IPN, and IDPN treated cells were exposed to a laser irradiance of 1.5 W/cm2 (808 nm, 8 min) for single photothermal therapy or chemo-photothermal therapy, and the IDPN treated cells not subjected to laser irradiation served as a single chemotherapy group. After incubation was done for another 24 h, the cells were washed three times and further incubated with 100 μL of a serum-free medium containing 10 μL of CCK8 for 45 min. The absorbance of each well was recorded at 450 nm using a microplate 13



reader (ELx808, BioTek Instruments). The relative cell viability was quantitatively calculated as a percentage of that of the control (non-treated cells). Meanwhile, the cell viability of HeLa cells was evaluated in the same way as described above. The biocompatibility of IPNs not exposed to laser irradiation was also detected using CCK8, by incubating them with NIH-3T3 and EMT-6 cells at concentrations of 0-300 μg/mL for 48 h. The cytotoxic effect of the various treatments on EMT-6 cells was also assessed by cell apoptosis analysis. Briefly, EMT-6 cells seeded in 6-well plates (8×105 cells per well) were treated with free ICG, IPNs, and IDPNs at the ICG concentration of 15 µg/mL (the corresponding Dox concentration was 3.9 µg/mL) for 12 h. The cells that were not subjected to any treatment were used as controls. Then, the medium was removed and replaced with fresh medium. Subsequently, the free ICG, IPN, and IDPN treated cells were exposed to a laser irradiance of 1.5 W/cm2 (808 nm, 8 min). IPNs plus laser or IDPNs plus laser was used for single photothermal therapy or chemophotothermal therapy, respectively. The IDPN treated cells not exposed to laser irradiation served as single chemotherapy group. After incubation was performed for another 24 h, the cells were trypsinized, harvested, and then resuspended in 100 µL binding buffer containing 5 µL of Annexin-V FITC and 5 µL of propidium iodide, using an Annexin V/PI cell apoptosis kit (BD, NJ, USA). Then, the mixture was incubated for 15 min in dark and another 400 µL of binding buffer was added to it before analysis was performed. Finally, the cell apoptosis levels were determined using a flow 14


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cytometer (FACS CANII, BD, USA). 2.8. Animals and tumor model All animal related experiments were conducted following the protocols approved by the institutional animal care committee of the University of Electronic Science and Technology of China (UESTC). The six-week-old female BALB/c mice or nude mice weighting about 20 g were supplied by the Laboratory Animal Centre of Sichuan University (Chengdu, China). To develop the EMT-6 tumor-bearing mouse model, the EMT-6 tumor cells (murine mammary cancer cells) were injected subcutaneously into the right back region of the mice at a concentration of ~2×106 cells/mouse. When the tumor reached a size of 80-100 mm3 (volume = length × width2/2), the mice were subsequently used for the following experiments.

2.9. In vivo fluorescence/photoacoustic imaging and biodistribution analysis Two BALB/c nude mice with tumor volumes of about 100 mm3 were intravenously injected via the tail vein with free ICG or IDPNs (100 μL, ICG concentration 150 μg/mL). At 2, 4, 8, and 24 h after administration, mice were anesthetized by the intraperitoneal injection of sodium pentobarbital, and the whole-body fluorescence imaging was performed by using IVIS® Lumina Series III (PerkinElmer, Inc., Waltham, MA, USA). The mice were sacrificed 24 h after they were injected and the tumors, hearts, livers, spleens, lungs, and kidneys were collected for ex vivo fluorescence imaging. The average fluorescence intensity of each organ and tumor was analyzed by the instrument software. 15



For in vivo photoacoustic (PA) imaging, two BALB/c nude mice with tumor volumes of about 100 mm3 were intravenously injected via the tail vein with free ICG or IDPNs (100 μL, ICG concentration 150 μg/mL). Then, a multispectral optical tomography system (MSOT inVision 128, iThera Medical, Germany) was used to acquire the PA images and PA signal intensity of the tumor sites 0, 1, 4, and 24 h after administration.

2.10. In vivo temperature measurement during laser irradiation Four BALB/c mice bearing EMT-6 tumors were intravenously injected with saline (as the control), free ICG (2 mg/kg ICG), IPNs (2 mg/kg ICG), and IDPNs (2 mg/kg ICG, 0.8 mg/kg Dox) through the tail vein. The tumors were irradiated using the 808 nm laser at 1.5 W/cm2 for 10 min. The infrared thermographic maps of the tumor tissues were obtained using a Ti27 infrared thermal imaging camera (Fluke, USA) during illumination (0, 2, 4, 6, 8, and 10 min).

2.11. In vivo therapeutic efficacy and biosafety of nanoparticles The BALB/c mice bearing EMT-6 tumors were randomly divided into the following five groups (5 mice per group) and underwent different treatments: (1) saline (control); (2) saline (control) with NIR laser irradiation; (3) free ICG (2 mg/kg ICG) with NIR laser irradiation; (4) IPNs (2 mg/kg ICG) with NIR laser irradiation; (5) IDPNs (2 mg/kg ICG, 0.8 mg/kg Dox); and (6) IDPNs (2 mg/kg ICG, 0.8 mg/kg Dox) with NIR laser irradiation. The saline, free ICG, and different nanoparticle formulations were administered via tail injection at days 0 and 3. For the laser treatment groups, the 16


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tumors of mice were irradiated using the 808 nm laser at 1.5 W/cm2 for 8 min, 24 h post-injection. Subsequently, the tumor sizes and body weights were measured every 3 days using vernier calipers and the electronic weighing scale during the treatment process. After 15 days, blood samples were collected from all groups of mice for the measurement of the biochemical markers of hepatic (ALT, AST, ALP, TP) and renal (BUN, CREA) functions as well as the complete blood count analysis (red blood cell, white blood cell, platelet, mean platelet volume, hematocrit, hemoglobin, mean corpuscular volume, the percentage of intermediate cells, and mean corpuscular hemoglobin concentration). Afterward, all the mice were sacrificed, and the tumors were excised, weighed, and photographed. The isolated tumor tissues and major organs were gathered and fixed in a 4% (w/v) paraformaldehyde solution and then embedded in paraffin blocks for 24 h, and sectioned at 5 mm. Then, the histopathology of tumor tissues and major organs as well as the proliferation of tumor cells were detected by H&E staining and Ki67 kits (Beyotime, China), respectively. Immunochemistry staining for testing Bax was also performed by using a Bax antibody staining kit (Abcam Inc., Cambridge, MA, USA). All the images were acquired using an optical microscope (BX53, Olympus, Japan). The sections were observed to obtain the proliferation index (percentage of Ki-67-positive cells from the total cells), by assessing three representative fields of immunolabeled tumor cell nuclei.

2.12. Intratumoral distribution of therapeutic nanoparticles The tumor frozen sections from the sacrificed mice were incubated with the rat anti-mouse CD31 and anti-HIF-1α primary antibodies (Abcam Inc., Cambridge, MA, 17



USA), which was followed by incubation with the Alexa Fluor 488-conjugated goat anti-mouse secondary antibody, used for the immunohistochemical (IHC) identification of tumor blood vessels and hypoxia, respectively. The cell nuclei were stained with DAPI. A fluorescence microscope (AxioImageA2, Zeiss, Germany) was used for imaging the tumor blood vessels/hypoxia and determining the location of IDPNs.

2.13. Statistical analysis All experiments were carried out in triplicates or using more specimens. Data were reported as mean ± standard deviation (SD) values. Statistical analysis was performed using the GraphPad Prism Software version 6.0 (GraphPad Software Inc., San Diego, CA, USA). The differences among groups were considered significant for * P

NIR-Light-Triggered Anticancer Strategy for Dual-Modality Imaging

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