Near-Infrared Fluorescent Dye-Decorated Nanocages to Form

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Near Infrared Fluorescent Dye-decorated Nanocages to Form Grenade-Like Nanoparticles with Dual Control Release for Photothermal Theranostics and Chemotherapy Chun-Yen Lin, and Ming-Jium Shieh Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00088 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 5, 2018

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Bioconjugate Chemistry

Near Infrared Fluorescent Dye-decorated Nanocages to Form Grenade-Like Nanoparticles with Dual Control Release for Photothermal Theranostics and Chemotherapy

Chun-Yen Lin† and Ming-Jium Shieh*,†,§ †

Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan §

Department of Oncology, National Taiwan University Hospital and College of Medicine, #7, Chung-Shan South Road, Taipei 100, Taiwan

*Ming-Jium Shieh, MD, PhD, Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan. E-mail: [email protected]; Tel: 886-2-23123456 ext 67142.

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ABSTRACT: Recently, nanoparticles (NPs) have been widely investigated for delivery of anticancer drugs. Here, a dual control drug-release modality was developed that uses naturally occurring protein apoferritin loaded with doxorubicin (DOX) and ADS-780 near infrared (NIR) fluorescent dye-decorated NPs (ADNIR NPs). ADNIR NPs act as a grenade to detonate the targeted tumor site following laser irradiation (photothermal therapy, PTT) and explode into cluster warheads (apoferritin-loaded DOX nanocages, AF-DOX NCs) that further destroy the tumor cells (chemotherapy). Light was shown to disrupt the grenade-like structure of NPs to release AF-DOX NCs as well as DOX from NCs in low-pH intercellular environments. In vitro and in vivo studies showed that the structure of AF-DOX NCs was disassembled to release DOX, which then killed the cancer cells in organelles with acidic environments. In vivo studies showed that the ADNIR NP-decorated with NIR dye facilitated tracking of the accumulated NPs at the tumor site using an IVIS imaging system. Overall, targeted ADNIR NPs with dual-release mechanisms were developed for use in photothermal theranostic and chemotherapy. This modality has high potential for application in cancer treatment and clinical translation for drug delivery and imaging.

KEYWORDS: Doxorubicin, Photosensitizer, Apoferritin, Cancer diagnosis, Colorectal cancer

Introduction Recent trends in cancer diagnosis and treatment have focused on non-invasive methods, such as high-intensity focused ultrasound and other approaches using magnetic force and light as suitable candidates for external stimuli.1-4 These external stimuli not only focus on the targeted regions but also affect the pathogenic microenvironments. For example, light-induced


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photothermal energy can increase the compartments of vascular endothelial cells to facilitate the penetration of nanoparticles (NPs) into the blood vessel in cancer therapy.5 The generation of heat can also disrupt the extracellular matrix surrounding the tumor cells, which is usually produced by carcinoma-associated fibroblasts, to achieve a physical contact with cytotoxic T lymphocytes and induce an immune response.6 In addition to external stimuli, internal stimuli such as pH, enzyme, and peroxidase can also be used to induce the release of NP drugs.7-10 Conventional photothermal therapy (PTT) may limit the activities of non-targeting photosensitizer molecules and damage the surrounding normal tissue. The use of exogenous chromophores is more selective for the release of drugs at targeted sites without causing damage to the normal tissue. Therefore, it is essential to select appropriate chromophores to specifically target pathological locations. Other studies have reported the use of exogenous materials in targeted nanotherapy such as metal oxides, carbon nanohorns, gold NPs, and organic chromophores, such as indocyanine green (ICG) tricarbocyanine near infrared (NIR) fluorescent dyes.11-14 Although, ICG has been approved by U.S. Food and Drug Administration (FDA), it lacks efficient heat generation, short bloodstream circulation half-life, and has low fluorescence intensity.15-17 Recent studies have investigated the efficacy of heptamethine dyes with similar structures, such as infrared (IR)-780, IR-786, IR-820, and IR-825,18-20 which can penetrate deeper into tissues, thereby increasing the quantum yield and decreasing the incidence of fluorophore photobleaching.21 However, the therapeutic effects of heptamethine dyes are limited by poor aqueous stability and short circulation half-life.22 The IR-780 iodide is a hydrophobic cationic dye that is more stable and has a higher quantum yield than ICG dye. The NIR dye ADS-780 has a similar structure to IR-780, but with the substitution of one methyl group with a hydroxyl group.23


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Carrier molecules are needed to overcome the hydrophobic properties of photosensitizer drugs to achieve efficient targeting. Typically, polymeric micelles, silica-based NPs, nanotubes, and nanogold are utilized as artificial carrier molecules.24-27 However, the use of such artificial carrier molecules mostly may impose immune response to the patient, manufacturing process complexity, and critical problems related to their biocompatibility have largely impeded the related developments.28-30 Compared with conventional carrier molecules, the application of protein NPs for drug delivery has continued to attract increasing attention over the last few years.31, 32 In fact, several protein cages composed of albumin, ferritin, transferrin, small heat shock proteins, and virus-like particles have been developed.33-36 Especially, Abraxane (an albumin-bound formulation of paclitaxel) was approved by FDA as a solvent-free formulation of paclitaxel for the treatment of metastatic breast cancer.37, 38 The hydrophobic drug becomes soluble once bound to albumin. In this study, apoferritin nanocages (AF NCs), which have a biologically appropriate inner diameter of 8 nm and outer diameter of 12 nm, were employed as ideal NPs for drug delivery. AF also contains specific binding sites for fatty acids and absorbed photosensitizers, as well as various metal ions (e.g., Cu2+, Au3+, and Pd2+) and photosensitizers (e.g., ZnF16Pc) with high loading capacity within the interior cavity.39-43 In addition, AF is a biocompatible molecule with a large surface area for multivalent conjugation, high biocompatibility, biodegradability, and pH sensitivity, which disassembles at pH 5.0 and reassembles at pH 7.4. Especially, the AF surface has active targets of transferrin receptor protein 1 (TfR1), which is overexpressed in human cancer cells.44-46 By exploiting this intrinsic tumor-targeting property, the use of such novel NCs may help achieve tumor-specific targeting without the need for additional targeting peptides or small molecular ligands. The enhanced


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permeability and retention (EPR) effect can also be used for passive targeting to accumulate more NPs at the tumor site.47 Here a dual control release NP system with external photothermal effects and inner cellular pH-triggered release was developed for photothermal theranostics and chemotherapy. As a brief explanation of the loading procedure, AF NCs were loaded with DOX using an assembly/disassembly method,48 and ADS-780 dye was decorated with a group of AF-DOX NCs (Figure 1A). ADNIR NPs act as a grenade to detonate the targeted tumor site following laser irradiation (photothermal therapy, PTT) and explode into cluster warheads (apoferritin-loaded DOX nanocages, AF-DOX NCs) that further destroy the tumor cells (chemotherapy) (Figure 1B). Injection of ADNIR NPs into the tail vein of mice resulted in passive accumulation of ADNIR NPs at the tumor site due to the EPR effect. Next, the tumor was irradiated with an 808-nm laser to disrupt ADNIR NPs. The released AF-DOX NCs were actively targeted to TfR1 expressed on the surfaces of the tumor cells and then taken up into the cells via receptor-mediated endocytosis. In the endosome and lysosome environments, the low pH induced DOX release from the disassembled AF to kill tumor cells (Figure 1C). Testing for cytotoxicity of PTT and chemotherapy showed that the viability of HT-29 cells was only 21.7% after 48 h. The effects of tumor inhibition were evaluated in a mice HT-29 tumor model. The results showed that photoirradiation significantly reduced the tumor size and had minimal effects on normal tissue because ADNIR NPs were confined to the photothermal regions. Overall, the results of the present study validated the use of these novel NPs with two released mechanisms for noninvasive targeted photothermal theranostics and chemotherapy. It is expected that this novel cancer treatment will be suitable for clinical application.


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Figure 1. Schematic mechanism of dual drug controlled release and the cancer treatment process of apoferritin loaded with doxorubicin (DOX) and ADS-780 near infrared (NIR) fluorescent dyedecorated NPs (ADNIR NPs). (A) Loading procedures of doxorubicin (DOX) and ADS-780 NIR dye. (B) ADNIR NPs act as a grenade to detonate the targeted tumor site following laser irradiation (PTT) and explode into cluster warheads (apoferritin-loaded DOX nanocages, AF-


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DOX NCs) that further destroy the tumor cells (chemotherapy). (C) Illustration of the mechanism of dual drug controlled release and the cancer treatment process of ADNIR NPs.

RESULTS AND DISCUSSION Characterization of ADNIR NPs. Dynamic light scattering (DLS) results showed that the average size of ADNIR NPs was 64.2 nm and the average polydispersity index was 0.133, which indicated that the prepared NCs were uniform and suitable for intravenous injection (Figure 2A). Analysis of the morphologies of AF NCs, AF-DOX NCs, ADNIR NPs and laser-irradiated ADNIR NPs under a TEM showed that the structure of ADNIR NPs was disrupted after exposure to 808-nm laser irradiation at 1.2 W/cm2 for 3 min. The results illustrated that the ADS780 molecules decorated the AF-DOX NC surface to form a homogenously self-assembled structure (ADNIR NPs) (Figure 2B). We proposed that the AF-DOX NCs absorbed ADS-780 NIR dye via electrostatic and hydrophobic interactions.49, 50 We observed the form of ADNIR NPs consisting of individual AF-DOX NCs assembled on the core. After NIR irradiation, the separated structure of ADNIR NPs was observed via TEM. Microscopic observation confirmed that AF and ADNIR NPs had an average size of 14.2 nm (n = 3) and 60.1 nm (n = 3), respectively.


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Figure 2. Characteristics of ADNIR NPs. (A) The size of the ADNIR NPs was measured using dynamic light scattering. (B) Transmission electron microscopy images of apoferritin nanocages (AF NCs), apoferritin-loaded doxorubicin (DOX) nanocages (AF-DOX NCs), apoferritin loaded with DOX and ADS-780 near infrared (NIR) fluorescent dye-decorated NPs (ADNIR NPs), and


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laser-irradiated ADNIR NPs. Yellow arrows indicate the location of ADNIR NPs. Scale bar = 100 nm.

To characterize the affinity between the hydrophobic NIR dye and protein nanocage, we used Autodock-Vina (version 1.1.2) and PyMOL (Version 2.0) software to simulate the mechanism by which the ADS-780 NIR dye absorbed onto the nanocage. The results demonstrated that the ADS-780 NIR dye docked onto the apoferritin surface (Figure S1). Furthermore, we utilized circular dichroism to examine whether the secondary structure of the nanocage was affected by NIR dye. Figure 3A reveals a characteristic peak at approximately 260 nm, and signals in the region from 250–270 nm are dominated by phenylalanine residues.51-54 When the loading content of ADS-780 NIR dye in nanoparticles increased, the peak intensity decreased, indicating that the secondary structure of nanocages might be influenced by ADS-780 NIR molecules. This result might be attributable to the interaction of ADS-780 molecules with the hydrophobic residue phenylalanine of nanocages, leading to a massive effect on the alpha helical structure of the nanocages.55-57 Moreover, we used a fluorescence spectrometer to confirm the assembly formation of ADNIR NPs. As shown in Figure 3B, the ADNIR NPs had no emission peak near 810 nm compared with NIR dye in DMSO, indicating that the fluorescence of dye was quenched in a tight packing of ADS-780 NIR dye molecules after being decorated with nanocages19, 58 and that the nanoparticle size was homogenous with a diameter of approximately 64.2 nm according to the DLS measurement. We then measured the fluorescent spectrum of ADNIR NPs after laser irradiation. Compared with NIR dye in DMSO, ADNIR NPs and laser-irradiated ADNIR had no peak in the fluorescence spectrum, indicating that ADNIR NPs and ADNIR NPs with laser irradiation resulted in an aggregation form and NIR dye was photobleaching due to laser


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irradiation. Hydrophilic free DOX in PBS and AF-DOX in PBS both exhibited fluorescence near 590 nm, but the ADNIR NPs in PBS and laser-irradiated ADNIR NPs had a shift to approximately 584 nm (Figure 3C).

Figure 3. (A) Circular dichroism spectra of AF-DOX NCs and ADNIR NPs with different D/P ratios. (B) The NIR fluorescence of ADS-780 NIR dye in PBS, ADS-780 NIR dye in DMSO, ADNIR NPs in PBS, and laser-irradiated ADNIR NPs in PBS. (C) The DOX fluorescence of free DOX in PBS, AF-DOX NCs in PBS, ADNIR NPs in PBS, and laser-irradiated ADNIR NPs in PBS.

DOX was physically loaded within AF using a pH-mediated disassembly and reassembly method. In brief, DOX was loaded into AF at different D/P ratios (5:1, 1:1, and 1:5) to form AFDOX NCs. A D/P ratio of 1:1 was chosen (encapsulation efficiency 48%) as an experimental template to further mix the ADS-780 NIR dye at different D/P ratios (1:6, 1:3, 1:1.5, and 1:0.5) to form ADNIR NPs. The cationic dye ADS-780 was absorbed onto the surface of the protein cages via electrostatic and hydrophobic interactions due to the AF cavity were occupied by DOX (Table 1). The optimal loading efficiencies of DOX and the ADS-780 NIR dye were 48% (D/P = 1:1) and 57.1% (D/P = 1:3), respectively.


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Table 1. Characteristics of AF NCs, AF-DOX NCs, ADNIR NPs, and laser-irradiated ADNIR NPs

a

Nanoparticles formulations

D/P ratioa

Size (nm)

Polydispersity index

Zeta potential (mV)

EEb,c (%)

AF NCs

-

18.1

0.259

−15.7

-

AF-DOX NCs

1/1

18.7

0.249

−10.2

48

ADNIR NPs

1/3

64.2

0.133

−4.9

57.1

ADNIR + Laser

-

178.8

0.604

−11

-

D/P ratio (AF-DOX) = weight of doxorubicin/weight of NCs. D/P ratio (ADNIR NPs) = weight

of ADS-780 NIR dye/weight of AF-DOX NCs. b

EE (%) = Doxorubicin encapsulation efficiency (AF-DOX, %) = (weight of DOX in the NCs

/weight of feeding DOX) × 100%. c

EE (%) = Decorated ADS-780 NIR dye efficiency = (weight of the AF-DOX NCs-decorated

ADS-780 NIR dye/weight of feeding ADS-780 NIR dye) × 100%.

Absorbance Spectrum and Stability of ADNIR NPs. The stability of ADNIR NP was evaluated, and the results showed that ADNIR NPs were very stable in PBS with no obvious change in size over a period of 14 days, as confirmed using DLS (Figure 4A). The zeta potential of ADNIR NPs was stable at −4 to −6 mV for 14 days (Figure 4B). Next, the zeta potential of NPs was measured during the formation of ADNIR NPs. The native AF NCs had a negative charge of −15.7 mV, which increased to −10.2 mV after loading of DOX. After adding the ADS780 NIR dye, the unique structure of ADNIR NPs had a charge of −4.9 mV (Figure 4C). In comparison to ADNIR NPs (−4.9 mV) without laser irradiation, the ADNIR NPs were toward more negative charge (−11 mV) due to the larger aggregated form of nanocages after laser irradiation (ADNIR NPs PDI = 0.133 vs. laser-irradiated ADNIR NPs = 0.604) (Table 1). To


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ensure a maximum NIR absorbance peak of ADNIR NPs, free DOX (100 μg/mL), free ADS-780 NIR dye (12.5 μg/mL in DMSO), AF NCs (500 μg/mL), AF-DOX NCs (100 μg/mL of DOX), and ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of the ADS-780 NIR dye) were prepared to examine the corresponding maximum absorbance peaks. The DOX and AF-DOX NCs had characteristic peaks at approximately 480 nm indicating the DOX was successfully encapsulated in the AF NCs. The maximum peak of ADNIR NPs in PBS shifted 20 nm, from 770 nm to 790 nm, compared with the free ADS-780 NIR dye in DMSO, indicating that the NIR dye interacted with nanocages. Therefore, the peak was suitable for NIR light irradiation to generate thermal energy, while the free DOX, AF-DOX NCs, and AF NCs groups had virtually no NIR absorption regions (Figure 4D).


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Figure 4. The stability and absorbance status of ADNIR NPs. (A) Dynamic light scattering measurements showed no significant change in the size of ADNIR NPs over a period of 14 days. (B) The zeta potential of ADNIR NPs over a period of 14 days. (C) The zeta potentials of the synthesis steps of AF NCs, AF-DOX NCs, and ADNIR NPs. (D) The distribution of absorbance peaks of free DOX in PBS, free ADS-780 NIR dye in DMSO, AF NCs, AF-DOX NCs, and ADNIR NPs in PBS.

Photothermal Efficiency and DOX Release from ADNIR NPs. Next, the heat generation ability of ADNIR NPs upon exposure to 808-nm laser irradiation was investigated. Free ADS780 NIR dye (12.5 μg/mL) was dissolved in DMSO, while AF-DOX NCs (100 μg/mL of DOX) and ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of the ADS-780 NIR dye) were dissolved in PBS. All solutions were subjected to 808-nm laser irradiation at 1.2 W/cm2 for 400 s. As shown in Figure 5A, the maximum temperature increased from 25°C to 48.8°C, which was sufficient to cause hyperthermia (>42°C) and subsequent damage to the cancer cells.59 To evaluate the effects of external stimuli in the form of photothermal energy and pH on ADNIR NPs, PBS solution was prepared at two different pH values (7.4 and 5.0) and combined with or without 808-nm laser irradiation (irradiation was performed for 3 min at 1.2 W/cm2 starting at 0.5 h [denoted by the red arrow]). At pH 7.4 without irradiation, the cumulative DOX had plateaued at 28% in 24 h. At pH 7.4 with light irradiation, the release of DOX had the same trend, but the release increased to 35.2% in 24 h, indicating that irradiation with the 808-nm laser affected the stability of ADNIR NPs and caused slight separation of the AF-DOX NC subunits. Additionally, the conformation of the channels may be changed during drug encapsulation and temperature might affect the release of DOX by altering the ferritin pore structure, as the channels have been demonstrated to be sensitive to the changes in temperature.60 On the other


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hand, at pH 5.0 with irradiation, DOX concentration was boost increased from 1 to 12 h (10%– 47.8%) due to the acidic pH environment and heat produced by exposure to 808-nm irradiation. The heat allowed the release of DOX from the cavities of AF-DOX NCs, and the nanocages disassembly released lager amounts of DOX in the acidic environment (Figure 5B). We further examined photothermal efficiency of the prepared ADNIR NPs with four different conjugate dose (the encapsulated NIR dye contents were 12.5, 6.25, 3.125, and 1.5625 μg/mL) with the fixed D/P ratio (1:3) and irradiation dose (1.2 W/cm2) and six different laser exposed dose (1.2, 1, 0.8, 0.6, 0.4, and 0.2 W/cm2) with the fixed D/P ratio (1:3) and NIR dye content (12.5 μg/mL). In different conjugate dose, the photothermal efficiency was positively correlated with the drug content of NIR dye. The higher NIR dye had higher photothermal effect in 400 s (Figure 5C). Meanwhile, the photothermal effect became weaker as the irradiation dose was decreased (Figure 5D).


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Figure 5. Trends in photothermal temperatures and cumulative drug-release profiles under different conditions. (A) The ADNIR NPs had a high absorbance to NIR light that caused the medium temperature to increase from 25°C to 48.8°C. (B) The cumulative effect of pH and laser light (irradiation was performed for 3 min at 1.2 W/cm2 starting at 0.5 h [denoted by the red arrow]) on drug release. (C) The photothermal efficiency of different drug content of NIR dye. (D) The photothermal efficiency of laser dose. Bars represent the means ± SD (n = 3).

Cellular Uptake and Competition Assay. Flow cytometry was used to measure cell TfR1 expression level in HT-29 colorectal cancer cell line. The TfR1 expression of HT-29 was 76%,


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which indicated that the HT-29 colorectal cancer cell was suitable for competition assay (Figure 6A). The binding affinity of FITC-labeled ADNIR NPs to HT-29 cells with high TfR1 expression level was investigated using confocal microscopy at 4°C for 1 h. The results showed that the FITC-labeled ADNIR NPs were bound to HT-29 cells (green color) with no energy supplement, indicating that ADNIR NPs retained the ability to bind to tumor cells (Figure 6B). To determine the photoirradiation effect on cellular uptake, HT-29 cells were incubated with ADNIR NPs at 37°C for 3 h, followed by exposure to 808-nm laser irradiation at 1.2 W/cm2 for 3 min, compared with no irradiation, and incubated for 24 and 48 h. As shown in Figure 6C, when exposed to 808-nm laser irradiation, the amount of DOX increased with time by 1.2- and 1.5-fold at 24 and 48 h, respectively. This finding suggested that the structure of NPs was disrupted by exposure to 808-nm laser irradiation and DOX was further released from the NCs. We proposed that the ADNIR NPs became unstable and disassembled into AF-DOX NCs due to laser irradiation. The increase of temperature loosened the structure of apoferritin and the encapsulated DOX leak out from nanocages.61 These results were in accordance with previous drug-release profile data showing that photoirradiation promoted the release of DOX from ADNIR NPs. Next, FITC was conjugated to the surfaces of ADNIR NPs, and the amount of FITC-labeled ADNIR NPs in HT-29 cells was measured to determine the specific targeting ability. HT-29 cells were pretreated with apoferritin (AF, 10 mg/mL) for 24 or 48 h to compete with FITC-labeled ADNIR NPs or not pretreated with AF. In the AF pretreatment group, the FITC-labeled ADNIR fluorescence was effectively suppressed by 2- and 1.6-fold at 24 and 48 h, respectively,


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compared with the findings in the absence of pretreatment. These results confirmed that ADNIR NPs had the ability to target the TfR1 molecules on the surfaces of HT-29 cells (Figure 6D). To further investigate the targeted competition ability of ADNIR NPs, flow cytometry was used to evaluate the ability of ADNIR NPs to target TfR1 after pretreatment with AF (10 mg/mL) as a competing molecule. As shown in Figure 6E, the green line (76% expression, without blocking with AF) shifted to pink (17% expression, with AF blocking), demonstrating a decrease in expression by 59%, which indicated that tumor uptake was mediated by TfR1–apoferritin interactions.

Figure 6. Transferrin receptor protein 1 (TfR1) expression level in HT-29 colorectal cancer cell line and examination of ADNIR NP-binding and uptake mechanisms. (A) TfR1 expression of


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HT-29 cells was measured using flow cytometry. (B) Binding ability of ADNIR NPs to HT-29 cells at 4°C for 1 h. The confocal microscopy images scale bar = 10 μm. (C) DOX was released from ADNIR NPs at 24 and 48 h in response to photoirradiation. (D) ADNIR NPs and apoferritin (AF) (10 mg/mL) cellular uptake competition test measured using a microplate reader. (E) ADNIR NPs and AF (10 mg/mL) cellular uptake competition assay determined using flow cytometry.

Photothermal Cytotoxicity of ADNIR NPs. First, the viability of HT-29 cells was determined after treatment with AF NCs (500 µg/mL), only 808-nm laser irradiation (1.2 W/cm2, 3 min), free ADS-780 NIR dye (6.25 and 12.5 µg/mL), free DOX (100 µg/mL), and AF-DOX NCs (100 μg/mL of DOX) (Figure 7A). AF, laser irradiation alone, and free ADS-780 NIR dye (6.25 and 12.5 µg/mL) caused little cytotoxicity to HT-29 cells (AF-treated cell viability, 91.1%; laser irradiation alone-treated cell viability, 90.5%; free ADS-780 NIR dye-treated cell viability, 90.2% in 6.25 µg/mL, and 92.3% in 12.5 µg/mL after 48 h). Free DOX was slightly more toxic than AF-DOX at equal amounts of DOX (Free DOX-treated cell viability, 51.3%; AF-DOXtreated cell viability, 55.6% after 48 h). Next, the cytotoxic effects of ADNIR NP chemotherapy and PTT were evaluated in vitro. The optimal concentrations of the ADS-780 NIR dye (6.25 or 12.5 μg/mL) and DOX (100 μg/mL) were used to treat HT-29 cells. The results showed that cell viability was significantly decreased with increasing concentrations of the ADS-780 NIR dye, from 6.25 to 12.5 μg/mL, whereas an increase in the duration of irradiation enhanced cytotoxicity (ADNIR NPs with light irradiation-treated cell viability, 57.1% in 30 sec; 50.6% in 60 sec; 45.4% in 120 sec; 36.3% in 180 sec, decorated with 6.25 μg/mL ADS-780 NIR dye)


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(ADNIR NPs with light irradiation-treated cell viability, 47.9% in 30 sec; 38.3% in 60 sec; 30.5% in 120 sec; 21.7% in 180 sec, decorated with 12.5 μg/mL ADS-780 NIR dye) (Figure 7B). To visualize laser irradiation-induced cytotoxicity in AF in PBS (control), 808-nm laser irradiation alone, AF-DOX NCs (100 μg/mL), and laser-irradiated ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye) groups by fluorescence microscopy, 1 µM calcein AM was used to stain live HT-29 cells (live cells are colored green). Microscopic analysis showed that there were no dead cells in the AF and only light irradiation groups, while there were many dead cells in the AF-DOX NCs group and especially in laser-irradiated ADNIR NPs group (fewer green fluorescent cells) (Figure 7C).

Figure 7. Cytotoxicity of live and dead cells determined using MTT cell proliferation assay and fluorescence microscopy. (A) The optimal treatment concentration of each drug was determined. There were significant differences in apoferritin (AF) between the phosphate-buffered saline (PBS; control) group and the DOX or AF-DOX NCs groups. (B) ADNIR NPs in HT-29 cells were treated for different irradiation times. There were significant differences between without laser-irradiated ADNIR NPs group and laser-irradiated ADNIR NPs group. (C) Fluorescence of live HT-29 cells added with AF in the PBS (control), 808-nm laser irradiation alone, AF-DOX


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NCs, and laser-irradiated ADNIR NPs groups. The fluorescence microscopy images scale bars = 100 μm. Bars represent the means ± SD (n = 3). *P < 0.05, and **P < 0.01.

The Location of ADNIR NPs in HT-29 cells. Confocal microscopy enabled tracking of the FITC-labeled ADNIR NPs. The fluorescence showed the locations of the FITC-labeled ADNIR NPs (green) and the release of DOX (red) at 24 and 48 h (Figure 8A). HT-29 cells showed greater accumulation of FITC-labeled ADNIR NPs and greater DOX release from NCs at 48 h than at 24 h. Also, DOX had entered into the cell nuclei. Quantification of fluorescence intensity showed an approximately 1.7-fold increase in DOX release from NCs and an approximately 2.8fold increase in cellular uptake of FITC-labeled ADNIR at 48 h compared with that at 24 h (Figure 8B).


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Figure 8. Tracking ADNIR NPs using confocal microscopy. (A) Confocal fluorescence images of HT-29 cells to locate ADNIR NPs. (B) The average fluorescence intensity was quantified using ImageJ software. Bars represent the means ± SD (n = 3). *P < 0.05, and **P < 0.01. The confocal microscopy images scale bar = 10 μm.

Pharmacokinetics of ADNIR NPs. We examined the pharmacokinetics of free DOX and DOX in ADNIR NPs. In brief, nude mice were separated into two groups (n = 5). The mice were intravenously injected with free DOX (10 mg/kg) or ADNIR NPs (injection dose of ADS-780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg). Blood samples were collected at different time points (0.5, 1, 3, 6, 12, and 24 h). The plasma DOX concentration was calculated by detecting the fluorescence intensity of DOX. As shown in Figure 9 and Table 2, the


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concentration of DOX in blood decreased faster than that of ADNIR NPs after intravenous injection, indicating that DOX has a shorter blood circulation time. T1/2 and CL (clearance) showed that ADNIR NPs had a longer circulation time than DOX (T1/2, 13.99 h vs. 9.51 h; CL, 273.85 mL/h/kg vs. 1146.91 mL/h/kg). In addition, the volumes of distribution for ADNIR NPs and DOX were 5527.38 and 15734.11 mL/kg, respectively. We assessed the biodistribution of tissue lysates from tumors and visceral organs to clarify the distribution of ADNIR NPs. Briefly, the FITC-labeled ADNIR NPs (injection dose of ADS-780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg) were injected into tail veins of nude mice bearing HT-29 tumors (n = 5) in 24 h before the first irradiation. The FITC fluorescence of ADNIR NPs was measured via HPLC and converted into protein concentrations by calculating a standard and normalizing the data to the organ weight. Apparently, the results demonstrated that ADNIR NPs preferentially accumulated in the tumors at 24 h before the first round of irradiation (Figure S2). The fluorescence of FITC-labeled ADNIR NPs was detected in the tumor, lung, and liver, particularly in the tumor, with low fluorescence signals in other organs.


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Figure 9. Pharmacokinetic analysis after a single injection.

Table 2. Pharmacokinetics of DOX in ADNIR NPs

a

Parametera

Unit

DOX

ADNIR NPs

T1/2

h

9.51

13.99

AUC0‑∞

μg·h/mL

6.04

11.58

Vd

mL/kg

15734.11

5527.38

CL

mL/h/kg

1146.91

273.85

MRT

h

13.72

20.18

T1/2, terminal half-life; AUC0‑∞, area under the concentration-time curve from 0 up to t based on

the sum of exponential terms; Vd, volume of distribution; CL, clearance, volume of plasma clearance of the nanocages; MRT, mean residence time.


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In Vivo Tumor Targeting with IVIS Spectrum System. ADNIR NPs have great potential for tumor imaging due to NIR absorbance. Tumor targeting was investigated in BALB/c nude mice bearing HT-29 tumors using IVIS spectrum in vivo imaging system. The treatment schedule is shown in Figure 10A. The red spots indicate high tumor accumulation of ADNIR NPs after PTT (Figure 10B). At 4 days, the IVIS image showed greater accumulation of ADNIR NPs at the tumor site, indicating the stromal components were destructed after exposure to 808-nm laser light. ADNIR NPs were retained at the tumor site at the last observation conducted on day 27, suggesting that the remaining free ADS-780 NIR dye of ADNIR NPs accumulated in the tumor after laser irradiation due to the high magnitude of mitochondrial membrane potential in tumor cells than normal cells.62, 63


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Figure 10. In vivo tumor targeting with ADNIR NPs evaluated using IVIS spectrum imaging. (A) ADNIR NPs were intravenously injected for photothermal theranostics and chemotherapy. Scheme of the in vivo treatment schedule. (B) The tumor-targeting effect was observed using IVIS spectrum imaging (the measurement unit for the image display is p/s/cm2/sr).

Effects of PTT and Temperature on Tumors. Observation of the mice with HT-29 tumors after intravenous injection and 808-nm laser irradiation (1.2 W/cm2, 15 min) showed that the top of tumor site had bruising of regions exposed to the 808-nm laser treatment for PTT due to accumulation of ADNIR NPs at the tumor site after intravenous injection for 1 day (Figure 11A). Further investigation showed that the tumor sites of the mice had encrusted wounds after day 4. Tumor growth was effectively inhibited until day 27, which then showed slight regrowth.


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Meanwhile, the increase in temperature at the tumor site during irradiation was monitored and imaged using a thermal camera system (Figure 11B). The highest photothermal temperature of ADNIR NPs reached 62.8°C in 15 min by irradiation at 1.2 W/cm2 for 15 min, whereas the temperature of AF-DOX NCs increased only to 41.8°C for 15 min due to the lack of ADS-780 NIR dye.

Figure 11. The effect of PTT and monitored tumor temperature of treatment with ADNIR NPs or AF-DOX NCs with 808-nm laser irradiation in vivo. (A) HT-29 tumor growth was inhibited by 808-nm laser irradiation. (B) The highest temperature (62.8°C) of the tumor after the first treatment with ADNIR NPs plus 808-nm laser irradiated regions was compared with treatment with AF-DOX NCs with no obvious increase in temperature (41.8°C).

In Vivo Therapeutic Efficacy of ADNIR NPs. Tumor suppression effects of different treatments were investigated in BALB/c nude mice bearing HT-29 tumors. There was no


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significant difference in tumor growth or size between the free DOX and control groups. However, there was a significant difference in tumor size between the ADNIR alone and control groups on day 27 (488.4 ± 106 vs. 981.8 ± 150.5 mm3), whereas chemotherapy alone was not sufficient to achieve long-term suppression of tumor growth. The laser-irradiated ADNIR NPs group had efficiency tumor growth inhibition on day 6 after laser irradiation (27.9 ± 2.16 mm3). Meanwhile, compared to other treatment groups, tumor growth efficiently inhibited by PTT and the addition of chemotherapy in the laser-irradiated ADNIR NPs group, which had a synergistic effect on day 27 (189.7 ± 29.3 mm3) (Figure 12A). Also, body weight was monitored as an indicator of therapy-induced toxicity (Figure 12B). There was no significant decrease in the body weights of the mice during the treatment period, indicating no obvious toxic side effects. Survival analysis showed that the ADNIR NP-treated with laser irradiation group had a longer survival probability (>60 days) than other groups (Figure 12C).


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Figure 12. In vivo chemotherapy and PTT antitumor therapeutic efficacy in mice bearing HT-29 tumors. (A) The tumor inhibition efficacy of mice bearing HT-29 tumors after different treatments. Bars represent the means ± SD (n = 5). *P < 0.05, and **P < 0.01. (B) Body weights of mice in different treatment groups. (C) Kaplan–Meier survival analysis of mice bearing HT-29 tumors in different treatment groups.

Toxicity to major organs was assessed in the ADNIR NPs, ADNIR NPs alone, free DOX, and control PBS treatment groups by staining tissue sections with H&E. The tumor histological sections showed greatly efficiency of antitumor effects in laser-irradiated ADNIR NPs group (Figure 13). The only chemotherapy ADNIR NP-treated alone was not sufficient to inhibit the


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growth of tumor. Compared with control, the free DOX group showed no obvious influence on tumor tissue sections due to the lack of a PTT effect and rapid drug elimination by bloodstream according to pharmaceutics kinetic. All treatment groups major organ sections of the heart, liver, spleen, lung, and kidney showed no obvious damage. These findings also suggested that administration of laser-irradiated ADNIR NPs could eliminate damage to the normal tissues while focusing the therapeutic effects on the targeted pathological regions.

Figure 13. Hematoxylin & eosin-stained images of major organs and tumor pathological regions. There was no significant change in damage to heart, liver, spleen, lung, and kidney tissues after treatment with ADNIR NPs with laser irradiation. Treatment of tumor tissues with ADNIR NPs with laser light caused significant PTT effects as observed using microscopy. Scale bars = 100 μm.

CONCLUSIONS


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Here a novel ADNIR NP treatment was developed based on AF NCs that had formed grenadelike nanoparticle. These ADNIR NPs had encapsulated chemotherapeutic agents and decorated with photosensitizers for combined cancer therapy, also equipped with imaging ability by using the NIR dye. The results showed that dual drug release of ADNIR NPs could be induced by 808nm laser irradiation, and the intrinsic pH-sensitive property of AF caused drug release in low-pH environments. Especially, these NPs exhibited TfR1 targeted ability which largely enhanced cancer treatment both in vitro and in vivo. Hence, this modality provides a promising naturebased targeted therapy using NPs to deliver chemotherapeutic agents and image-guided theranostics for PTT. It has a high potential for application in cancer treatment and clinical translation for drug delivery and imaging.

EXPERIMENTAL PROCEDURES Materials and Cell Culture. HT-29 colorectal cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin– streptomycin in an incubator at 37°C with 5% CO2 under fully humidified conditions. Penicillin– streptomycin, DMEM, FBS, and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersburg, MD, USA). All other solvents were of analytical grade. Apoferritin (AF), RIPA buffer, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The dimethyl sulfoxide (DMSO) was obtained from Tedia (Fairfield, OH, USA). The calcein AM was obtained from Invitrogen Corporation (Carlsbad, CA, USA). The 0.1-μm syringe filter and Amicon ultra 50K filters were obtained from Merck Millipore (Billerica, MA, USA). The TfR1 antibody was obtained from GeneTex, Inc. (Irvine, CA, USA).


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Chemotherapeutic drug doxorubicin hydrochloride (DOX) was obtained from American Dye Source (Baie-d'Urfé, Quebec, Canada) and the ADS780HO NIR dye was synthesized by American Dye Source. Synthesis of AF-DOX NCs and ADNIR NPs. First, DOX was loaded into AF using an assembly and disassembly method, as previously reported.48 In brief, pH of the AF-containing solution was adjusted to 4 before the addition of DOX (D/P ratios, the weight concentration of drug / the weight concentration of protein ratio, of 5:1, 1:1, and 1:5). After 30 min, the pH was adjusted back to 7.4 overnight. The unloaded free DOX was removed by ultra-filtration using Amicon ultra 50K filters. To obtain ADNIR NPs, the ADS-780 NIR dye was dissolved in dimethyl sulfoxide (DMSO). Next, AF-DOX NCs were mixed in the solution at a D/P ratio of 1:6, 1:3, 1:1.5, or 1:0.5. Afterward, the solution was centrifuged at 2000 rpm for 10 min to remove large aggregates and then filtered through a 0.1-μm syringe filter. The structure of ADNIR NPs contained the AF-DOX NCs as its sphere core, and ADS-780 dye were decorated the surface of ADNIR NPs. The free ADS-780 NIR dye (DMSO ≤ 5%) was removed by ultrafiltration using an Amicon ultra 50K filter. Morphology, Particle Size and the Zeta-Potential of the Prepared Nanoparticles. The morphologies of AF NCs, AF-DOX NCs, ADNIR NPs, and laser-irradiated ADNIR NPs were observed under a transmission electron microscope (TEM; model H-7650 and model H-7100; Hitachi Corporation, Tokyo, Japan). NCs and NPs were negatively stained by 2% uranyl acetate. NC and NP sizes were measured by dynamic light scattering (DLS) using Zetasizer Nano ZS90 system (Malvern Instruments, Ltd., Malvern, England). All experiments were performed in triplicate, and all values are presented as the mean ± standard deviation (SD) (n = 3). Also, the maximum absorbance spectra peaks of free DOX (100 μg/mL), AF NCs (500 μg/mL), free ADS-


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780 NIR dye (12.5 μg/mL in DMSO), and ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye) were determined using a spectrophotometer with the materials in a quartz cell. Circular Dichroism (CD) Analysis. CD analysis of samples with various D/P ratios was performed on a J-815 spectropolarimeter (Jasco, Oklahoma City, OK, USA) using a quartz cylindrical cuvette with a path length of 0.1 mm. The spectra were examined via continuous wavelength scans over the range of 250–650 nm Loading Capacity. The DOX loading capacity (%) was calculated as the weight of DOX encapsulated in NCs divided by the total amount of added DOX. The efficiency of the decorated ADS-780 NIR dye (%) was calculated as the weight of the decorated ADS-780 NIR dye divided by the total weight of the added ADS-780 NIR dye. Different D/P ratios were followed for DOX and AF (5:1, 1:1, and 1:5). After formation of AF-DOX NCs, D/P ratios of the ADS-780 NIR dye to AF-DOX NCs were 1:6, 1:3, 1:1.5 and 1:0.5. Temperature Measured. A thermocouple was used to measure the medium temperature at every 40 to 400 s. First, 200 μL of ADNIR NPs were added to each well of 96-well plates containing DMEM medium at room temperature and then irradiated with a 808-nm laser (1.2 W/cm2). AF NCs, AF-DOX NCs (100 μg/mL of DOX) and ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye) were dissolved in phosphate-buffered saline (PBS). The different samples were exposed to the same irradiation energy and time intervals. Temperature was measured using a thermocouple needle (copper–constantan thermocouple; Omega Engineering, Stamford, CT, USA) connected to a data acquisition system (National Instruments Corporation, Austin, TX, USA). All experiments were repeated three times, and the values are presented as the mean ± SD.


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Drug Release Profile of ADNIR NPs. To examine the effects of 808-nm laser irradiation and pH on ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye), ADNIR NPs were prepared at two pH values (5.0 and 7.4) in PBS buffer combined with or without 808-nm laser irradiation at 1.2 W/cm2 for 3 min starting at 0.5 h and rotating at 37°C. At different time points (1, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h), 100 μL of the ADNIR NPs solution was collected into wells of 96-well plates. Then, absorbance was measured at 490 nm using a Spectramax plate reader (Molecular Devices, Sunnyvale, CA, USA). Flow Cytometry. Flow cytometry was used to measure the expression of TfR1 by TfR1 antibody, also called CD-71 antibody, in HT-29 cells. The cells (5 × 105 cells) were seeded into the wells of 6-well plates containing mediums and were cultured overnight. Afterward, the cells were trypsinized and collected into 15-mL centrifuge tubes. Then, 3 mL of the appropriate medium was added to each tube and was centrifuged for 15 min at 1500 rpm to remove the trypsin. The cell number in the suspensions was adjusted to 5 × 106 cells/mL. Then, 1 mL of PBS containing the TfR1 antibody (1:500) was added in the dark to target the cell surface. After 1 h cultured in 37°C, the cells were washed three times to remove any unbound antibody and transferred to new tubes containing 1 mL of PBS. The cell suspensions were immediately analyzed using flow cytometry (Becton Dickinson Biosciences, San Jose, CA, USA). Cellular Uptake and Competition Assay. HT-29 cells were pre-seeded at 5 × 105 cells into each well of 6-well plates and incubated for 24 h. ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye) were added to each well. HT-29 cells and ADNIR NPs were incubated at 37°C for 3 h. Afterward, the wells were with or without 808-nm laser irradiation (1.2 W/cm2, 3 min) and the plates were further incubated for 24 and 48 h to observe DOX cellular uptake. DOX fluorescence in the wells of plates was measured at 590 nm using a


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Spectramax plate reader. The final DOX fluorescence was normalized to the total amount of cell protein. In competition assay, we labeled fluorescein isothiocyanate (FITC) with ADNIR NPs (ADNIR to FITC dye ratio = 1:5). After ultra-filtration using an Amicon ultra 50K filter, HT-29 cells were incubated with the FITC-labeled ADNIR NPs with or without pretreatment with 10 mg/mL of AF for 1 h. Following pretreatment, removing the unbound AF by PBS washing three times, FITC-labeled ADNIR NPs were added into the wells of 6-well plates, which were then incubated for 24 and 48 h. Next, RIPA buffer was added to disrupt the cell walls, and the cell solution was centrifuged at 1500 rpm for 5 min to remove cellular debris. Finally, FITC fluorescence emission was measured at 525 nm using a Spectramax plate reader (Molecular Devices, Sunnyvale, CA, USA). All experiments were repeated three times, and the values are presented as mean ± SD. In Vitro Cytotoxicity and PTT. The cytotoxicity of various treatment groups and different 808-nm laser irradiation times was examined using HT-29 cells. In brief, HT-29 cells were seeded into the wells of 96-well plates at a concentration of 5 × 105 cells and incubated for 24 h. Then, the cells were treated with a culture medium containing ADNIR NPs (100 μg/mL of DOX and 6.25 μg/mL and 12.5 μg/mL of ADS-780 NIR dye). After 3 h, the medium was replaced with the fresh medium and the cells were incubated for an additional 48 h to further determine the photothermal efficacy (cytotoxicity) of 808-nm laser irradiation (1.2 W/cm2) for different time periods (30, 60, 120, and 180 s) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] cell proliferation assay. All experiments were repeated three times, and the values are presented as the mean ± SD. To investigate the phototherapeutic efficacy of ADNIR NPs, live cells were stained with calcein AM. Briefly, 5 × 105 HT-29 cells were seeded into the wells of 6-well plates and cultured


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overnight. Then, AF in PBS, AF-DOX NCs, and ADNIR NPs were added to the wells and the plates were irradiated. After 3 h at 37°C, the medium was replaced with the fresh medium or exposed to the 808-nm laser (1.2 W/cm2, 3 min) depending on the conditions and cultured for 48 h. Next, 1 μM calcein AM solution was added to each well, and incubated the cells at room temperature for 40 min. Finally, the wells were washed with PBS and immediately observed under a fluorescence microscope. The Location of ADNIR NPs in HT-29 cells. HT-29 cells (5 × 105 cells per well) were seeded into the wells of 6-well plates containing glass cover slides inside the wells and cultured overnight. After 3 h, the medium was replaced with fresh medium containing FITC-labeled ADNIR NPs (100 μg/mL of DOX and 12.5 μg/mL of ADS-780 NIR dye). After 24 and 48 h, the cells were washed three times with PBS. Cellular uptake and release of the drug were observed under a Zeiss LSM 780 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) at different time points (24 and 48 h). In the confocal image, the cell nuclei stained with 4',6-diamidino-2phenylindole (DAPI) appeared blue in color; ADNIR NP was labeled with green fluorescence, and DOX was appeared in red color. Pharmacokinetics Analysis. The nude mice were separated into two groups (n = 5). Mice were intravenously injected with free DOX (10 mg/kg) or ADNIR NPs (injection dose of ADS780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg). Submandibular blood samples were collected at different intervals (0.5, 1, 3, 6, 12, and 24 h), and whole blood samples were collected into EDTA-containing blood collection tubes. Next, the obtained samples were centrifuged at 3500 rpm for 10 min. DOX was extracted using a mixture of water and acetonitrile (50:50, v/v), and its plasma concentration was analyzed via HPLC.


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In Vivo Imaging and Thermal Imaging. When the tumor size increased to >55 mm3, the biodistribution of ADNIR NPs (injection dose of ADS-780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg) were studied using injections to the tail veins of mice bearing HT-29 tumors and imaged at 0, 1, 4, 6, 12, and 27 days after injection using IVIS imaging system (Xenogen Corporation, Alameda, CA, USA). The measurement unit for the image display is p/s/cm2/sr. In vivo, the distribution of thermal energy in the tumor was monitored using a thermographic camera (NEC Avio Infrared Technologies Co., Ltd., Tokyo, Japan). The mice were treated with ADNIR NPs and AF-DOX NCs and then irradiated at 1.2 W/cm2 for 15 min. In Vivo Tumor Inhibition. This study was approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC). Four-week-old, female BALB/c nu/nu mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and randomly assigned to one of the following four groups: a saline-treated control group, a free DOX group (10 mg/kg), an ADNIR NPs without 808-nm laser irradiation group (injection dose of ADS-780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg), and an ADNIR NPs with 808-nm laser irradiation group (injection dose of ADS-780 equivalent to 6 mg/kg and injection dose of DOX equivalent to 5 mg/kg of DOX). The tumors were irradiated with an 808-nm laser at 1.2 W/cm2 for 15 min. Treatment was started when the tumor grew to a size > 55 mm3. The first day (day 0), mice in the ADNIR NPs group received an intravenous injection through the tail vein, followed by irradiation with an 808-nm laser at 1.2 W/cm2 for 15 min at 24 h after intravenous injection. On day 3, mice in the ADNIR NPs group received second intravenous injection through the tail vein. On day 4, photoirradiation was applied a second time after a second injection of the ADNIR NPs.


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Mice in the ADNIR NPs alone group and a free DOX group received twice intravenous injection with no photoirradiation as the same time points of the ADNIR NPs group. In the control group, the mice were injected with PBS but received no photoirradiation as the same time points of the other groups. The tumor volume was determined using a caliper according to the formula, tumor volume = length × (width)2/2. The tumor volumes and body weights of the mice were measured every 3 days for 27 days, unless the tumor size had grown to > 1,000 mm3 or 15% weight loss occurred, which was designated as endpoints. We performed survival analysis using MedCalc statistical software (Version 17.9.7). Immunohistochemical Analysis. The major organs and tumor were excised, fixed in formalin for 1 week, embedded in paraffin, and then sliced to a thickness of 5 mm, followed by staining with hematoxylin and eosin (H&E) and examination under a microscope. Statistical Analysis. All data were analyzed using the Student’s t-test. Values of *P < 0.05 or **P < 0.01 were taken as indicating statistically significant differences between treatments. Bars represent the mean ± SD.

ASSOCIATED CONTENT Supporting Information Docking; ex vivo biodistribution. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: 886-2-23123456 ext 67142. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology, R.O.C. (MOST 102-2221-E-002-037-MY3).

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Near-Infrared Fluorescent Dye-Decorated Nanocages to Form

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