Hypotoxic and Rapidly Metabolic PEG-PCL-C3-ICG Nanoparticles for

Aug 31, 2017 - Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University,...
0 downloads 9 Views 3MB Size
Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

Hypotoxic and Rapidly Metabolic PEG-PCL-C3-ICG Nanoparticles for Fluorescence-Guided Photothermal/Photodynamic Therapy against OSCC Shuangshuang Ren, Xiao Cheng, Mengkun Chen, Chao Liu, Peichen Zhao, Wei Huang, Jian He, Zhengyang Zhou, and Leiying Miao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09522 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16

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

ACS Applied Materials & Interfaces

Hypotoxic and Rapidly Metabolic PEG-PCL-C3-ICG Nanoparticles for Fluorescence-Guided Photothermal/Photodynamic Therapy against OSCC Shuangshuang Ren,ξ Xiao Cheng,‡ Mengkun Chen,‡ Chao Liu,† Peichen Zhao,a Wei Huang,a Jian He,*,# Zhengyang Zhou,*,# and Leiying Miao *,† †

Department of Cariology and Endodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210093, Jiangsu, P. R. China.

ξ

Department of Periodontology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210093, Jiangsu, P. R. China.

#

Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University, Jiangsu, 210008, P. R. China. ‡

Collaborative Innovation Center of Chemistry for Life Sciences, College of Engineering and Applied Sciences, Nanjing University, Jiangsu, 210093, P. R. China. a

State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, 210093, P. R. China.

ABSTRACT: The development of agents for non-invasive photothermal/photodynamic therapies (PTT/PDT) against cancer remains challenging because most PTT agents cause side effects on normal tissues due to their high cytotoxicity and slow metabolism rate. We successfully synthesized an organic compound (C3), encapsulated in PEG-PCL with indocyanine green (ICG), to form hybrid nanoparticles (PEG-PCL-C3-ICG NPs) for use as a new PPT/PDT agent to treat cancer with a single irradiation. Compared with conventional PPT agents, such as Au nanorods, C3 showed better photothermal conversion stability, lower cytotoxicity and a faster metabolic rate, ensuring promising PTT efficacy in eliminating tumors during in vivo application, while ICG was used as a PDT agent. With 808-nm laser irradiation at tumor sites, the PEG-PCL-C3-ICG NPs were able to simultaneously produce hyperthermia through C3 and produce reactive oxygen species as well as a fluorescence-guided effect through ICG to kill oral squamous cell carcinoma (OSCC) cells. The combination of these hypotoxic and metabolic hybrid nanoparticles with radiation therapy has potential for the future treatment of OSCC.

KEYWORDS: C3, indocyanine green, photothermal therapy (PTT), photodynamic therapy (PDT), nanoparticles, OSCC

1. INTRODUCTION Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck mucosa and accounts for 90% of malignant tumors of the oral cavity.1 OSCC is a growing global problem, especially in South and Southeast Asia,2 where the vast majority of oral cavity tumor patients are diagnosed with OSCC.3 Currently, the standard therapy for OSCC includes surgical excision and a combination of chemotherapy and radiothera-

py. Additional, chemotherapeutic treatments may be provided to treat patients with confirmed distant metastases or to sensitize malignant cells to radiation.4 However, the 5-year survival rates have remained low for the past three decades. High mortality and significant toxicity caused by current strategies used in treating OSCC underline the urgent need for enhanced treatment options.5 Photothermal therapy (PTT) and photodynamic therapy (PDT) are considered effective alternatives for precancerous and cancerous oral lesions due to their

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

noninvasive nature and minimal cumulative side effects, especially PDT, which has been shown to be especially effective with negligible scar formation, even after repetitive treatments.6 PTT agents show promise in killing cancer cells due to the transformation of absorbed energy into heat, and PTT, as a new invasive treatment technique, has been extensively studied in the ablation of cancer cells.7-17 Gold-, copper-, and tungsten-based NPs are the most common PTT agents due to their strong absorbency under NIR.18-25 Unfortunately, most metal NPs have high toxicity and are poorly metabolized, which results in immune responses and damaged normal tissues, thereby limiting their clinical application.26 Owing to their rapid biodegradation and strong absorption spectra in NIR, organic nanomaterials have become popular alternatives, and potential applications of PTT in killing tumors have been recently reported.27,28 Here, we successfully synthesized an organic compound (C3) with excellent photothermal properties due to the presence of benzene rings and conjugated double bonds. Compared with conventional PTT agents, such as Au nanorods, C3 features better photothermal conversion stability, lower cytotoxicity and a rapid metabolic rate, ensuring promising PTT efficacy in eliminating tumors and better suitability for in vivo applications. In this study, C3 was equipped with indocyanine green (ICG) into PEG-PCL to form hybrid nanoparticles (PEG-PCLC3-ICG NPs). The PEG-PCL-C3-ICG NPs were able to simultaneously serve as PTT and PDT agents under 808nm laser irradiation. In vitro and in vivo tests showed that fluorescence-guided PTT/PDT rendered by the PEG-PCL-C3-ICG NPs under 808-nm laser irradiation holds promise as a cancer treatment. Hematology assays indicated that PEG-PCL-C3-ICG NP-assisted PTT/PDT combined treatment had no obvious in vivo cytotoxicity and ensured good biocompatibility. We believe these PEG-PCL-C3-ICG NPs have promise in clinical applications. 2. EXPERIMENTAL SECTION 2.1 Materials and animals. 3,4,9,10-Perylene diacid anhydride, pyridine, 2-ethylhexylamine, hydrochloric acid, mPEG-OH (Mw: 3500 Da) were obtained from the JenKem Company (Beijing, China), and ε-caprolactone (ε-CL), indocyanine green (ICG), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Page 2 of 16

(MTT), and 2,7-duchlorodihydrofluorscein diacetate (H2DCFDA) were obtained from Sigma Aldrich (St. Louis, MO). All other chemicals were available from J&K Chemicals (Beijing, China), and no further treatments were performed for direct use. HSCs were obtained from Fudan University (Shanghai, China). 2.2 Synthesis of Compound 1.29 A solution of 3,4,9,10-perylene diacid anhydride (3.82 g, 10.0 mmol) was dissolved in pyridine (100 mL), and 2ethylhexylamine (6.46 g, 50 mmol) was added. The reaction was stirred under Ar atmosphere at 120 °C for 24 h. The reaction solution was poured into an aqueous solution of hydrochloric acid (1 M, 1 L), and the precipitate was collected by filtration and dried under heating at 60 °C to give compound 1 in a yield of 4.72 g, (78.4 %) without any further purification. Mp: > 250 °C. 1H NMR (400 MHz, CDCl3) δ ppm: 8.58-8.61 (d, 4H, J = 5.0 Hz), 8.48-8.50 (d, 4H, J = 5.0 Hz), 4.11-4.15 (m, 4H), 1.96 (s, 2H), 0.89-1.40 (m, 28H). 2.3 Synthesis of Compound 2.29 Under Ar atmosphere, compound 1 (3.01 g, 5.0 mmol) was dissolved in DCM (100 mL). After being heated to reflux, bromine (20 mL) was added dropwise to the above solution. The mixture was refluxed for 3d. After being cooled to room temperature, the mixture was washed with saturated Na2S2O3 solution. The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The residue was purified with column chromatography to give red solid in a yield of 3.17 g (82.1%) Mp: > 250°C. 1H NMR (400 MHz, CDCl3) δ ppm: 9.36-9.38 (d, 2H, J = 8.0 Hz), 8.82 (s, 2H), 8.588.60 (d, 2H, J = 8.0 Hz), 4.11-4.16 (m, 4H), 1.93 (s, 2H), 1.41-1.32 (m, 16H), 0.96-0.87 (m, 12H). 2.4 Synthesis of Compound 3 illustrated in Scheme 1. Under Ar atmosphere, a toluene solution (15 mL) of compound 2 (850 mg, 1.1 mmol), polythiophene trimethyltin derivative (625 mg, 1 mmol), and Pd(PPh3)4 (35 mg, 0.03 mmol) was stirred at 120 °C for 36 h, and the toluene was removed under reduced pressure to give brown solid. The solid was purified with column chromatography in a yield of 421 mg (37 %). 1H NMR (400 MHz, CDCl3) δ ppm: 9.48 (br, 1H), 8.89 (br, 1H), 8.68 (br, 3H,), 8.31 (br, 3H), 7.28 (br, 2H), 4.14 (br, 6H), 2.55 (br, 6H). IR (KBr pellets, cm-1): 3402, 3131, 1669, 1661, 1400. Elemental analysis of C3 was calculated based on the molecular formula (C72H82N2O4S3)n as C: 76.14; H: 7.29; N: 2.46, S: 8.47 %. Found: C: 76.28; H: 7.36; N:

ACS Paragon Plus Environment

Page 3 of 16

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

ACS Applied Materials & Interfaces

Scheme 1. Synthesis of Compound 3 (C3). 2.59, S: 8.55 %. The average molecular weight (Mn) of C3 was ~4.8 kDa (Figure S7). 2.5 Synthesis of PEG-PCL. The mixture, which consists of a certain amount of ε-CL and m-PEG-OH, was placed in a vacuum-sealed tube and stirred at 160 °C for 24 h. To remove the unreacted monomer and oligomer after completion of polymerization, DCM was utilized to dissolve crude copolymers, and enough cold ethyl ether was used to let the product precipitate. The precipitates were filtered and dried via reduced pressure. The average molecular weight (Mn) of PEG-PCL was ~11.6 kDa (Figure S7).

chromatography (GPC) was used to measure the molecular weight and polydispersity of C3 and PEG-PCL. The number-average molecular weights (Mn), weightaverage molecular weights (Mw) and polydispersity index (PDI, Mw/Mn) were determined using polystyrene as the reference. Measurements were carried out using a Waters 515 HPLC pump, a Waters 2414 differential refractome-ter and three Waters Styragel columns (HT2, HT3 and HT4) with THF as the eluent at a flow rate of 1.0 ml min-1 at a temperature of 35 °C. The morphologies of PEG-PCL, PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3-ICG were examined using transmission electron microscopy (TEM, JEOL TEM-100). A spectrophotometer (UV3100, Shimadzu, Japan) was used to determine the UV-Vis absorption spectra of PEG-PCL, C3, ICG, PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCLC3-ICG. The size distribution and zeta potentials were assessed using dynamic light scattering (DLS) (BI9000AT, Brookhaven, USA). The fluorescence of PEGPCL-ICG, and PEG-PCL-C3-ICG were detected on a Fluoromax-4 spectrofluorometer (HoribaScientific, Edison, Nanjing). To study the photothermal and photodynamic effects, an 808-nm diode laser (LE-LS-8083000TFC-D, Leo Photonics Company, Shanghai) was used. To calculate the drug loading content and encapsulation efficiency of PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3-ICG, calibration curves for C3 and ICG obtained from absorbances at different concentrations (UV3100, Shimadzu, Japan) were employed. Drug loading content 

2.6 Synthesis of PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3-ICG. Scheme S1 shows the synthesis PEG-PCL-C3-ICG. Briefly, PEG-PCL (50 mg) was dissolved in deionized water (50 mL). A dichloromethane suspension (5 mL) containing C3 or ICG or C3/ICG (10 mg) was added to the above suspension after 5 min of sonication. The mixture was further treated with sonication for 30 min to collect the PEG-PCL-C3, PEG-PCL-ICG and PEG-PCL-C3-ICG by centrifugation, and the residue was removed by washing constantly five times with deionized water. 2.7 Characterization. 1H NMR and 13C NMR were performed on a Bruker AV400 spectrometer with CDCl3 as the solvent and tetramethylsilane as the internal standard. Fourier transform infrared spectroscopy (FTIR) was performed through mixing lyophilized dry powder of samples with KBr (5 mg: 1g, W/W) on a Bruker IFS 66V vacuum-type spectrometer. Gel permeation

Weight of the drug in NPs  100% Weight of the NPs

Encapsulation efficiency 

Weight of the drug in NPs  100% Weight of the feeding drug

2.8 Detection of ROS generated in cells. ROS generated in PEG-PCL-C3-ICG NPs-containing HSCs after NIR light irradiation were detected using an H2DCFDA probe. HSCs were seeded on a 6-well plate (5×103 cells per well) for 24 h and subsequently incubated with 200 µL PEG-PCL-C3-ICG (300 µg/mL) for 24 h. The positive control cells contained 200 µL H2O2 (75 mM). Additionally, 50 µL H2DCFDA (10 mM in DMSO) was added to the cells and incubated for another 1 h at 37 °C. The cells were washed three times with PBS and exposed to an 808-nm laser (2 W/cm2) for 5 min. The generated ROS were subsequently monitored using confocal laser scanning microscopy with an excitation of 488 nm and emission of 520 nm. ROS generated in cells, as in-

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 4 of 16

dicated by the H2DCFDA probe, were also quantitatively measured by flow cytometry (Cytomics FC 500 MCL, Beckman Coulter).

ter (UV3100, Shimadzu, Japan), respectively. UV absorbency was normalized to the tissue weight and presented as a percentage of dose/g.

2.9 In vitro cytotoxicity assay. The cytotoxicity of PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3-ICG to HSCs was determined by MTT colorimetric assay. Initially, HSCs were seeded on 96-well plates (104 cells/well) in 100 mL of Iscove’s modified Dulbecco's medium and incubated in 5% CO2 atmosphere at 37 °C overnight. The medium in each well was replaced with 100 mL fresh medium containing various concentrations of PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3ICG NPs (each concentration was examined in five replicates), and the medium was aspirated 24-h (with or without exposure to laser irradiation at an energy density of 2 W/cm2 for 3 min) and 48-h post-incubation. At the same time, the cells were washed twice with phosphatebuffered saline (PBS), followed by the addition of 20 mL of MTT solution (2.5 mg/mL in PBS) and subsequent incubation for another 4 h at 37 °C. The medium was aspirated, the collected cells were resuspended in 200 mL DMSO, and the absorbance of each well at 490 nm was measured using an iMark Enzyme mark instrument (BIO-RAD Inc., USA).

2.10 In vivo antitumor effect. One hundred microliters of HSC suspension containing 106 cells were injected subcutaneously into the left axillary lymph nodes of nude mice. The mice were randomly divided into four groups when tumor sizes reached approximately 500 mm3: a saline group, a PEG-PCL-C3 group, a PEGPCL-ICG group and a PEG-PCL-C3-ICG group. Each group consisted of seven mice. NIR laser radiation (808nm, 2 W/cm2) was carried out for 10 min at 8 h after the intravenous injection of PEG-PCL-C3, PEG-PCL-ICG, and PEG-PCL-C3-ICG (50 mg/kg). The body weight, tumor volume and survival of each mouse were observed and recorded. After 15 days, the mice were sacrificed, and the tumors were collected, weighed, washed three times with PBS and fixed in 10% neutral buffered formalin solution. Hematoxylin and eosin (H&E) staining and TUNEL apoptosis staining of the tumors were performed by the Nanjing Pharmaceutical Company and observed via fluorescence microscopy (IX71, Olympus).

2.10 In vivo NIR fluorescence imaging. HSC suspensions with a density of 106/mL in 100 µL PBS were injected subcutaneously into the left axillary lymph nodes of nude mice. The mice were kept for two weeks and provided food and water ad libitum. The PEG-PCLC3-ICG suspension (50 mg/kg) was injected into the tumor-bearing mice through the tail veins and imaged using the Maestro in vivo fluorescence imaging system (CRi Inc., Woburn, MA). The mice were anesthetized with isoflurane and scans were performed at 1 h, 4 h, 8 h, 12 h, 24 h, and 48 h post-injection. 2.11 Bio-distribution of ICG and C3 in tumorbearing mice. PEG-PCL-C3-ICG suspensions (50 mg/kg) were injected into tumor-bearing mice intravenously, and the mice were sacrificed (for each time point n = 5) 1 h, 4 h, 8 h, 12 h and 24 h after injection. The tumor, blood, heart, liver, spleen, lung and kidney were excised and weighed. Dimethyl sulfoxide (DMSO) was used to extract ICG and C3 from the organs. ICG and C3 were separated via gel silica column chromatography. ICG and C3 contents were calculated based on ICG and C3 calibration curves obtained using a spectrophotome-

2.11 Pathology analysis. We performed a time course of histological changes in the mice organs (heart, liver, spleen, lung, and kidney) 48 h after the PEG-PCL-C3ICG NP-based PTT/PDT treatment. The organs collected from the mice were embedded in paraffin after immobilization in 4% paraformaldehyde at 4 °C for 4 h, and then the sections were stained with hematoxylin and eosin (H&E) and observed using a light microscope, and representative images were taken. 2.12 Hematology and biochemical assay. Mouse blood was collected via extirpating with the naked eye at 1 day, 7 days and 21 days post-treatment for hematology and biochemical assays. To evaluate the biocompatibility and in vivo toxicity of PEG-PCL-C3-ICG NPs assisted with PTT/PDT treatment, serological liver function was analyzed by alanine aminotransferase (ALT), total bilirubin level (TBIL) and total protein (TP), kidney function was detected by blood urea creatinine (CRE) and nitrogen (BUN), and spleen function was correlated with the platelet level. White blood cells (WBC), Red blood cells (RBC), Lymphocytes, Monocytes (MON) and Neutrophils (NEU) were counted by flow cytometry assay, and levels of hemoglobin (HB) were used to check the immune response and potential cytotoxic effect.

ACS Paragon Plus Environment

Page 5 of 16

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

ACS Applied Materials & Interfaces

Figure 1. (A) TEM image of PEG-PCL NPs and solution of PEG-PCL NPs. (B) TEM image of PEG-PCL-C3 NPs and solution of PEG-PCL-C3 NPs. (C) TEM image of PEG-PCL-ICG NPs and solution of PEG-PCL-ICG NPs. (D) TEM image of PEG-PCL-C3-ICG NPs and solution of PEG-PCL-C3-ICG NPs.

3. RESULTS AND DISCUSSION 3.1 Characterization. C3 was successfully synthesized and verified by 1H NMR (Scheme 1 and Figure S1-7), where the successful introduction of oligothiophene unit in C3 can be evidenced by two new peaks at 2.55 ppm corresponding to the protons of double β-substituted alkyl chains. Transmission electron microscopy (TEM) (Figure 1) was employed to investigate the morphology of the PEG-PCL, PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NPs. As displayed in Figure 1A, the PEG-PCL NPs showed a homogeneously dispersed spherical morphology with a 40-nm mean diameter. Nevertheless, the C3, ICG or C3 and ICG loads enhanced the mean diameters, and the PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NPs all exhibited 50-nm mean diameters (Figure 1B, C, and D). As shown in Table S1, we also observed that the drug loading content, encapsulation efficiency, diameter and zeta potential for the PEG-PCL-ICG, PEG-PCL-C3 and PEGPCL-C3-ICG NPs were very similar. UV-Vis spectra of PEG-PCL showed no apparent absorption in the NIR region (Figure 2A, B, and C), while a typical absorption

peak at 495 nm and a continuous high absorption intensity as a function of wavelength up to the NIR region was observed in C3 (Figure 2A). Furthermore, ICG had a strong absorption peak at 795 nm (Figure 2B). After assembly and separation via three rounds of centrifugation, new peaks corresponding to C3 and ICG appeared, indicating that C3 and ICG were successfully loaded (Figure 2A, B, and C). Due to the strong NIR absorbance of the PEG-PCL-ICG, PEG-PCL-C3 and PEGPCL-C3-ICG NPs, we next investigated the potency of these nanoparticles for translating the absorbed energy into heat. Based on the temperature curves of saline, the PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NPs are shown in Figure 2D. When exposed to 808-nm laser irradiation for 10 min, the temperatures of the PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NPs solutions increased from 25 °C to 39 °C, 45 °C and 47 °C, respectively, whereas the control solution only showed a slight temperature increase. These data confirmed that the PEG-PCL-ICG and PEG-PCL-C3 nanoparticles exhibited photothermal properties and that synergistic photothermal heating effects for cancer therapy were achieved when C3 and ICG were successfully

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 6 of 16

Figure 2. (A) UV-vis spectra of PEG-PCL dissolved in distilled water, C3 dissolved in dichloromethane and PEGPCL-C3 dissolved in distilled water; (B) UV-vis spectra of PEG-PCL dissolved in distilled water, ICG dissolved in dichloromethane and PEG-PCL-ICG dissolved in distilled water; (C) UV-vis spectra of PEG-PCL dissolved in distilled water, PEG-PCL-ICG dissolved in distilled water, PEG-PCL-C3 dissolved in distilled water and PEG-PCL-C3ICG dissolved in distilled water; (D) Temperature-time curve of saline, PEG-PCL-ICG dissolved in distilled water, PEG-PCL-C3 dissolved in distilled water and PEG-PCL-C3-ICG dissolved in distilled water. All the concentrations are l mg/mL.

loaded on the PEG-PCL-C3-ICG conjugates. Au nanorods were used as a PTT agent due to a superior photothermal potency and low cytotoxicity.30 The photothermal conversion, stability and cytotoxicity of equivalent Au nanorods and PEG-PCL-C3-ICD NPs were detected and compared for potential in vivo applications. Five cycles of NIR laser irradiation (0.5 W/cm2, 808-nm laser, 10 min)/cooling (1 h) were performed. As indicated in Figure 3A, five cycles later, elevation of temperature of PEG-PCL-C3-ICG NPs decreased little compared to the Au nanorods even though the first elevation of temperature in PEG-PCL-C3-ICG NPs was lower than that in Au nanorods. The PEG-PCL-C3-ICG NPs featured a

better photothermal conversion stability for C3 was amorphous and was not destroyed by hyperthermia, while Au Nanorod is club-shaped. Hyperthermia will induce Au nanorod deformation from club-shaped to spherical-shaped particles.31 Based on the cytotoxicity results (Figure 3B), we concluded that the PEG-PCLC3-ICG NPs featured lower cytotoxicity at 24 h or 48 h incubation at the same concentration. Taken together, PEG-PCL-C3-ICD NPs were more suitable for in vivo applications. 3.2 In vitro cytotoxicity. As displayed in Figure 4, the MTT assay was performed to assess and compare the in vitro cytotoxicity of the PTT effect, rendered by C3, and

ACS Paragon Plus Environment

Page 7 of 16

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

ACS Applied Materials & Interfaces

Figure 3. (A) Temperature of Au nanorod and PEG-PCL-C3-ICG NP solutions (both 1 mg/mL) after five cycles of NIR laser irradiation (2 W/cm2, 808-nm laser, 5 min)/cooling down (1 h). (B) In vitro cell viability of Au nanorod and PEG-PCL-C3-ICG NP solutions (both 1 mg/mL) at 24 h or 48 h post-incubation. Error bars represent standard deviations of four independent experiments. ** p < 0.01.

the PDT effect, provided by ICG. HSCs were cultured with PEG-PCL-ICG NPs (24 h), PEG-PCL-C3 NPs (24h) and PEG-PCL-C3-ICG NPs (24 h or 48 h) and irradiated by NIR laser (808-nm, 2 W/cm2) for 3 min. As shown in Figure 4, none of the samples displayed obvious cytotoxicity without irradiation, regardless of incubation duration. In contrast, cells exposed to NIR irradiation featured positive dose-dependent cytotoxicity. PEG-PCL-C3-ICG NPs up-taken by HSCs cells were subjected to 808-nm laser irradiation (PTT/PDT) and showed the highest cytotoxicity. In particular, cell viability was 32.5% in the PEG-PCL-C3-ICG NP group (PTT/PDT) at C3 and ICG concentrations of 20 µg/mL, whereas the cell viability was 72.5% in the PEG-PCLC3 NP group (PTT) and 65.7% in the PEG-PCL-ICG NP group (PDT) at C3 or ICG concentrations of 20 µg/mL. Collectively, these results clearly showed that the PEGPCL-C3-ICG NPs maintained photothermal and photodynamic functions and had synergistic effects against HSCs. 3.3 In vitro ROS detection at single-cell level. Given their high cytotoxicity, reactive oxygen species (ROS) kill cancer cells when selectively generated in tumors. To clarify whether sufficient ROS can be produced by PEG- PCL-C3-ICG NPs under laser irradiation, HSCs were incubated with PEG-PCL-C3-ICG NPs and irradiated with an 808-nm laser (2 W/cm2) for 5 min. At the same time, another group treated with H2O2 was used as

a positive control, and those treated with laser irradiation alone or PEG-PCL-C3-ICG NPs without irradiation served as negative controls. Visible intracellular ROS generation was assessed by staining cells with H2DCFDA, a standard probe to detect ROS, followed by CLSM and flow cytometry measurements. As shown in Figure 5A, in groups where HSCs were treated with either 50 mM H2O2 or PEG-PCL-C3-ICG NPs combined with NIR irradiation, an intensive green fluorescence signal was detected, indicating ROS generation. In the negative controls with cells treated by laser irradiation only and PEG-PCL-C3-ICG NPs without irradiation, no significant detectable signals were observed. As shown in Figure 5B, we used flow cytometry to evaluate the quantification of induced ROS intensity. In terms of intracellular fluorescence intensity, PEG-PCL-C3-ICG NPs combined with laser irradiation was higher than that of the positive control (H2O2) and 2.8 times higher than that of the negative controls. These data confirmed that abundant ROS can be selectively generated in cells through the uptake of PEG-PCL-C3-ICG NPs and NIR irradiation, which means that light-triggered PEG-PCLC3-ICG NPs are suitable candidates in PDT. 3.4 NIR fluorescence imaging and the biodistribution of ICG and C3. Before the in vivo NIR fluorescence imaging, the in vitro fluorescence of PEGPCL-ICG and PEG-PCL-C3-ICG were monitored to verify whether C3 would quench the fluorescence of

7

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 8 of 16

Figure 4. (A) In vitro cell viability of PEG-PCL-C3-ICG NPs at different concentrations after 24 h or 48 h incubation without laser irradiation. (B) In vitro cell viability of PEG-PCL-C3 NPs at different concentrations after 24 h incubation with laser irradiation. (C) In vitro cell viability of PEG-PCL-ICG NPs at different concentrations after 24 h incubation with laser irradiation. (D) In vitro cell viability of PEG-PCL-C3-ICG NPs at different concentrations after 24 h incubation with laser irradiation. Error bars represent standard deviations of four independent experiments. * p < 0.05, ** p < 0.01.

ICG. As displayed in Figure S8, we could observe the fluorescence of ICG in PEG-PCL-C3-ICG NPs and PEG-PCL -ICG NPs was very close and fluorescence of ICG in PEG-PCL-C3-ICG NPs wouldn’t be quenched by C3. After injecting NPs via the tail vein, tumorbearing mice were visualized using a non-invasive and real-time NIR fluorescence imaging technique to study the in vivo tissue distribution of PEG-PCL-C3-ICG NPs. The time-lapse NIR images of tumor-bearing mice postinjection are shown in Figure 6A. The different fluores-

cence intensities of ICG are displayed using different colors and decreases in a descending sequence of red, orange, yellow, green, and blue. 1 h post-injection, a noticeable fluorescence signal was observed in liver (Figure 6A) and no signal detected in the tumor, indicating that most of the PEG-PCL-C3-ICG NPs were rapidly recognized by the reticuloendothelial system (RES). However, 4 h and 8 h post the injection, strong fluorescence signals were detected in the tumor areas, proving that PEG-PCL-C3-ICG NPs could be passively enriched

8

ACS Paragon Plus Environment

Page 9 of 16

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

ACS Applied Materials & Interfaces

Figure 5. (A) CLSM images of singlet oxygen generation in HSCs following various treatments as indicated. Green: ROS indicator H2DCFDA; scale bar, 50 µm. (B) Quantification of induced ROS intensity achieved by flow cytometry.

Figure 6. (A) Fluorescence imaging of tumor-bearing mice following i.v. injection of PEG-PCL-C3-ICG NPs (50 mg/kg). Excitation and emission of ICG in the NIR region was 808 nm and 820 nm, respectively. Tumor sites are marked with yellow circles. (B) Bio-distribution analysis of ICG. (C) Bio-distribution analysis of C3.

at tumor locations due to the enhanced permeability and retention (EPR) effect. With the time lapsed, stronger fluorescence signals were detected in tumor areas,

whereas negligible fluorescence signals were detected in the liver. The EPR effect means that tumor tissue has a vascular system with holes of pore size 20-500 nm.32-34

9

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 10 of 16

Figure 7. (A) Tumor volume of HSC-tumor-bearing mice receiving different treatments (dose, 50 mg/ kg) as indicated. Data are presented as the means ± SD (n = 7). * p < 0.05 and ** p < 0.01. (B) Survival curves of HSC-tumor-bearing mice receiving different treatments. (C) Histological observation of tumor tissue after different treatments. Tumor sections were stained with hematoxylin and eosin (H&E). (D) Detection of cell apoptosis in tumor tissues after treatment by staining tumor sections with deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and observation with optical microscope. Scale bar, 50 µm.

The size of PEG-PCL-C3-ICG NPs was approximately 50 nm (Figure 1D), so PEG-PCL-C3-ICG NPs could take advantage of the EPR effect, accounting for the stronger fluorescence signal in the tumor area. The escape of PEG-PCL-C3-ICG NPs from the capture of RES and the entry of PEG-PCL-C3-ICG NPs at the tumor sites may explain the weaker fluorescence signal detected in the liver.

To further examine the bio-distribution of ICG delivered by PEG-PCL-C3-ICG NPs in vivo, organs extracted from sacrificed mice were collected at different time points post-injection. We extracted ICG content from various tissues and expressed it as percentage of dose per unit mass of tissue. As shown in Figure 6B, a significant accumulation of ICG in tumors was observed. At 8 h post-injection, the ICG content in tumors reached a maximum of approximately 6.75% dose/g tissues, indicating thatPEG-PCL-C3-ICG NPs have a quick EPR

10

ACS Paragon Plus Environment

Page 11 of 16

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

ACS Applied Materials & Interfaces

Figure 8. (A) Serum bio-chemical test of alanine aminotransferase (ALT), total bilirubin (TBIL), total protein (TP). (B) Creatinine (CRE), blood urea nitrogen (BUN), and hematology assay of platelets (PLT). (C) Red blood cells (RBC), hemoglobin (HB), and white blood cells (WBC). (D) Monocyte (MON), lymphocyte (LYM), and neutrophils (NEU). Level 1, 7, and 21 days post-treatment of PEG-PCL-C3-ICG rendered PTT/PDT.

accumulation, enabling the delivery of ICG to tumors at relatively higher concentrations. The bio-distribution of C3 delivered by PEG-PCL-C3-ICG NPs in vivo is shown in Figure 6C. Extremely similar C3 biodistributions and contents were observed in all tissues, indicating that ICG and C3 could be selectively delivered to tumor sites with high accumulations based on the EPR effect; furthermore, both compounds featured similar metabolism rates. In most PTT/PDT systems, the PTT agent metabolizes slowly when compared with the PDT agent. The PTT agent is usually heavy-metalbased, which typically has slow metabolic rates.26 We thus anticipated that the PEG- PCL-C3-ICG NPs would show relatively low toxicity in vivo, offering broad appeal for clinical applications.

3.5 In vivo anti-tumor efficacy. In this study, HSC tumor-bearing mice were used as animal models to evaluate the efficacy of PEG-PCL-C3-ICG NPs in PTT/PDT treatment. Mouse samples were divided into four groups with seven mice per group, each of which had an average cell growth rate of 500 mm3. A-saline-treated group served as the control group, whereas the three other groups were intravenously injected with PEG-PCL-ICG NPs, PEG-PCL-C3 NPs, and PEG-PCL-C3-ICG NPs. All groups injected with NPs were exposed to NIR laser irradiation (808-nm, 2 W/cm2, 8 min) 8 h after intravenous injection. As indicated in Figure 7A, the saline group showed rapid tumor growth, and tumor volume increased 13-fold on day 15 when compared with day 1. While the PEG-PCL-ICG NPs and the PEG-PCL-C3 NPs provided sufficient PDT and PTT, respectively, for controlling tumor growth in the first 5 days, these effects

11

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

were only observed on a recurring cycle. In contrast, the PEG-PCL-C3-ICG NPs played an active role in continuously suppressing tumor volume. As a result, by the end of the test, tumor volumes increased approximately 2.3-fold, 2.6-fold, and 1.1-fold compared with initial volumes at day 1 for the PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NP groups, respectively, indicating synergistic therapeutic effects on the ablation of solid tumors. Figure 7B shows the survival curves of the mice; the life span of mice in the saline group was no more than 43 days due to a rapid tumor growth rate, while in the groups intravenously injected with PEGPCL-ICG NPs or PEG-PCL-C3 NPs, 3 or 4 mice died within 60 days, respectively. In contrast, the PEG-PCLC3-ICG NP group displayed the longest survival span, and only one mouse died, indicating that the combined PTT/PDT treatment rendered by the PEG-PCL-C3-ICG NPs had significantly superior efficacy in prolonging the lifetime of mice compared with PDT or PTT alone. The body weight of the mice was also determined, and no obvious changes in the body weight of the mice were observed during the course of the study for all groups (Figure S9), implying that PEG-PCL-C3-ICG NPs displayed no obvious acute toxicity. The therapeutic efficacy in all groups was further assessed by hematoxylin and eosin (HE) staining and deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Tumor tissues in the PEG-PCL-ICG, PEG-PCL-C3 and PEG-PCL-C3-ICG NP groups presented large ranges of necrosis and apoptosis. However, the degree of necrosis and apoptosis was the largest in the PEG-PCL-C3-ICG NP group (Figure 7B, C). Therefore, we concluded that the combined PTT/PDT therapy of PEG-PCL-C3-ICG NPs provided the best therapeutic effect on tumor necrosis and apoptosis and had significant superiority in preventing tumor growth and prolonging the life span of mice. 3.6 Safety evaluation. Heart, liver, spleen, lung, and kidney were harvested and weighed to evaluate the in vivo safety. As indicated in Figure S10A, the mice treated with PEG-PCL-C3-ICG did not show any noticeable variation in the mass ratio of the organ to the body. Figure S10B shows representative H&E staining pictures of these organs treated with saline or PEG-PCL-C3-ICG NPs assisted with PTT/PDT treatment. Compared to images from saline group, no apparent necrosis, hy-

Page 12 of 16

dropic degeneration, pulmonary fibrosis, histological abnormalities and inflammation lesions were seen in any organs from the mice treated with the PEG-PCL-C3ICG NPs assisted PTT/PDT treatment, which provides solid evidence of the good biocompatibility of these PEG-PCL-C3-ICG NPs in vivo. Serum bio-chemical and hematology studies were performed to estimate the potential cytotoxicity of the PEG-PCL-C3-ICG NPs on the body after PTT/PDT treatment. Serum levels of alanine aminotransferase (ALT), total bilirubin (TBIL), and total protein (TP) were used to assess liver function (Figure 8A), whereas serum levels of blood urea nitrogen (BUN), platelets (PLT), and creatinine (CRE) were used to evaluate kidney and spleen function (Figure 8B). Negligible changes in statistical significance were observed when compared with untreated mice (control group), revealing that the PEG-PCL-C3-ICG NP-based PTT/PDT treatment did not breakdown metabolism. Hematotoxicity is concerned with the application of nanomaterials in vivo. As displayed in Figure 8C, red blood cell (RBC), white blood cell (WBC) and hemoglobin (HB) counts with respect to hematopoietic and aerobic capacity were similar throughout the course of treatment when compared with untreated mice (control group). Peripheral blood lymphocyte (LYM), monocyte (MON) and neutrophil (NEU) levels were determined to analyze potential immune responses (Figure 8D). Negligible changes in statistical significance were observed throughout the course of treatment when compared with untreated mice (control group), indicating the potential for using PEG-PCL-C3-ICG NPs in clinical applications. 4. CONCLUSION In summary, PEG-PCL-C3-ICG NPs were successfully prepared to serve as agents in combined PTT and PDT tumor treatments under 808-nm laser irradiation. Based on in vitro and in vivo experiments, we found that C3 can be used as a new PTT agent due to its enhanced photothermal conversion stability, lower cytotoxicity and higher metabolic rate compared with previously reported PTT agents (e.g., Au nanorods). In the PEG-PCL-C3ICG NPs, C3 and ICG were able to absorb NIR light to induce local hyperthermia and generate ROS, respectively. PEG-PCL NPs were used to prevent instability and rapid ICG clearance, which enhanced PDT treatment. The combined PEG-PCL-C3-ICG NPs had ad-

12

ACS Paragon Plus Environment

Page 13 of 16

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

ACS Applied Materials & Interfaces

vanced properties when compared with the PEG-PCLC3 and PEG-PCL-ICG NPs. In vitro studies showed that the PEG-PCL-C3-ICG NPs had better biocompatibility than Au at low doses. Additionally, similar conclusions were drawn from in vivo anti-OSCC efficiency tests. When treated with PEG-PCL-C3-ICG NPs during PTT/PDT, significantly better therapeutic effects were observed than those observed for PTT or PDT alone. Thus, these PEG-PCL-C3-ICG NPs may be used in the fluorescence-guided photothermal/photodynamic therapy against OSCC.

REFERENCES (1) Chinn, S. B.; Myers, J. N. Oral Cavity Carcinoma: Current Management, Controversies, and Future Directions. J. Clin. Oncol. 2015, 33, 3269-3276. (2) Rao, S. V. K.; Mejia, G.; Roberts-Thomson, K.; Logan, R. Epidemiology of Oral Cancer in Asia in the Past Decade-an Update (2000-2012). Asian Pac. J. Cancer Prev. 2013, 14, 5567-5577. (3) Warnakulasuriya, S. Global Epidemiology of Oral and Oropharyngeal Cancer. Oral Oncol. 2009, 45, 309316.

ASSOCIATED CONTENT Electronic Supplementary Information (ESI) available: Scheme of the synthesis of PEG-PCL-C3-ICG. 1HNMR spectra of compound 1, compound 2, compound 3 (C3). 13 CNMR spectra of compound 2, compound 3 (C3). IR spectra compound 3 (C3). Gel permeation chromatography (GPC) of C3 and PEG-PCL. Drug loading content, encapsulation efficiency, diameter and zeta potential for PEG-PCL-ICG NPs, PEG-PCL-C3 NPs and PEG-PCL-C3-ICG NPs. Fluorescence spectrum of PEGPCL-ICG and PEG-PCL-C3-ICG NPs. The whole mouse weight and coefficients of major organs. AUTHOR INFORMATION Corresponding Authors:

(4) Thomas, G. J. Oral Squamous Cell Carcinoma. Am. J. Surg. Pathol. 2008, 29, 167-178. (5) Economopoulou, P.; Perisanidis, C.; Giotakis, E. I.; Psyrri, A. The Emerging Role of Imunotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC): Anti-Tumor Immunity and Clinical Applications. Ann. Transl. Med. 2016, 4, 173-185. (6) Lecaros, R. L.; Huang, L.; Lee, T. C.; Hsu, Y. C. Nanoparticle Delivered VEGF-A siRNA Enhances Photodynamic Therapy for Head and Neck Cancer Treatment. Mol. Ther. 2016, 24, 106-116. (7) Pan, L.; Liu, J.; Shi, J. Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 15952-15961.

*E-mail: [email protected]; *E-mail: [email protected]; *E-mail: [email protected]. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work is supported by Natural Science Foundation of China (No. 81570952, No. 51472115), Jiangsu Province Natural Science Foundation of China (BK20161114) and Medical Science and technology development Foundation, Nanjing Department of Health (No. ZKX16054).

(8) Kuang, Y.; Zhang, K.; Cao, Y.; Chen, X.; Wang, K.; Liu, M.; Pei, R. Hydrophobic IR-780 Dye Encapsulated in cRGD-Conjugated Solid Lipid Nanoparticles for NIR Imaging-Guided Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 12217-12226. (9) Peng, J.; Dong, M.; Ran, B.; Li, W.; Hao, Y.; Yang, Q.; Tan, L.; Shi, K.; Qian, Z. "One-for-AII"-Type, Biodegradable Prussian Blue/Manganese Dioxide Hybrid Nanocrystal for Trimodal Imaging-Guided Photothermal Therapy and Oxygen Regulation of Breast Cancer. ACS Appl. Mater. Interfaces 2017, 9, 13875-13886. (10) Cai, W.; Gao, H.; Chu, C.; Wang, X.; Wang, J.; Zhang, P.; Lin, G.; Li, W.; Liu, G.; Chen, X. Engineering

13

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Phototheranostic Nanoscale Metal-Organic Frameworks for Multimodal Imaging-Guided Cancer Therapy. ACS Appl. Mater. Interfaces 2017, 9, 2040-2051; (11) Dou, Y.; Yang, X.; Yang, W.; Guo, Y.; Wu, M.; Liu, Y.; Li, X.; Zhang, X.; Chang, J. PB@Au CoreSatellite Multifunctional Nanotheranostics for Magnetic Resonance and Computed Tomography Imaging in Vivo and Synergetic Photothermal and Radiosensitive Therapy. ACS Appl. Mater. Interfaces 2017, 9, 1263-1272. (12) Pei, H.; Zuo, X.-L.; Zhu, D.; Huang, Q.; Fan, C.H. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550-559. (13) Li, J.; Fan, C.-H.; Pei, H.; Shi, J.-Y.; Huang, Q. Smart Drug Delivery Nanocarriers with Self-Assembled DNA Nanostructures. Adv. Mater. 2013, 25, 4386-4396. (14) Yao, G.-B.; Li, J.; Chao, J.; Pei, H.; Liu, H. J.; Zhao, Y.; Shi, J.-Y.; Huang, Q.; Wang, L.-H.; Huang, W.; Fan, C.-H. Gold-Nanoparticle-Mediated Jigsaw-Puzzlelike Assembly of Supersized Plasmonic DNA Origami. Angew. Chem. Int. Ed. 2015, 54, 1-5. (15) Li, J.; Pei, H.; Zhu, B.; Liang, L.; Wei, M.; He, Y.; Chen, N.; Li, D.; Huang, Q.; Fan, C.-H. SelfAssembled Multivalent DNA Nanostructures for Noninvasive Intracellular Delivery of Immunostimulatory CpG Oligonucleotides. ACS Nano 2011, 5, 8783-8789. (16) Pei, H.; Liang, L.; Yao, G.-B.; Li, J.; Huang, Q.; Fan, C.-H. Reconfigurable Three-Dimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angew. Chem. Int. Ed. 2012, 51, 9020-9024. (17) Chen, L.-Z.; Chao, J.; Qu, X.-M.; Zhang, H.-B.; Zhu, D.; Su, S.; Aldalbahi, A.; Wang, L.-H.; Pei, H. Probing Cellular Molecules with PolyA-Based Engineered Aptamer Nanobeacon. ACS Appl. Mater. Interfaces 2017, 9, 8014-8020. (18) Wang, X.-X.; Cao, D.-W.; Tang, X.-J.; Yang, J.J.; Jiang, D.-Y.; Liu, M.; He, N.-Y.; and Wang, Z.-F. Coating Carbon Nanosphere with Patchy Gold for Production of Highly Efficient Photothermal Agent. ACS Appl. Mater. Interfaces 2016, 8, 19321-19332. (19) Shanmugam, V.; Chien, Y.-H.; Cheng, Y.-S.; Liu, T.-Y.; Huang, C.-C.; Su, C.-H.; Chen, Y.-S.; Kumar, U.;

Page 14 of 16

Hsu, H.-F.; Yeh, C.-S. Oligonucleotides-Assembled Au Nanorod-Assisted Cancer Photothermal Ablation and Combination Chemotherapy with Targeted Dual-Drug Delivery of Doxorubicin and Cisplatin Prodrug. ACS Appl. Mater. Interfaces 2014, 6, 4382-4393. (20) Chen, L.-J.; Sun, S.-K.; Wang, Y.; Yang, C.-X.; Wu, S.-Q.; Yan, X.-P. Activatable Multifunctional Persistent Luminescence Nanoparticle/Copper Sulfide Nanoprobe for in Vivo Luminescence ImagingGuided Photothermal Therapy. ACS Appl. Mater. Interfaces 2016, 8, 32667−32674. (21) Solorzano, I. O. D.; Prieto, M.; Gracia Mendoza, G.; Alejo, T.; Irusta, S.; Sebastian, V.; Arruebo, M. Microfluidic Synthesis and Biological Evaluation of Photothermal Biodegradable Copper Sulfide Nanoparticles. ACS Appl. Mater. Interfaces 2016, 8, 21545−21554. (22) Kim, Y. K.; Kang, E. B.; Kim, S. M.; Park, C. P.; In, I.; Park, S. Y. Performance of NIR-Mediated Antibacterial Continuous Flow Microreactors Prepared by Mussel-Inspired Immobilization of Cs0.33WO3 Photothermal Agents. ACS Appl. Mater. Interfaces 2017, 9, 3192-3200. (23) Dong, K.; Liu, Z.; Liu, J.-H.; Huang, S.; Li, Z.H.; Yuan, Q.-H.; Ren, J.-S.; Qu, X.-G. Biocompatible and High-Performance Amino Acids-Capped MnWO4 Nanocasting as a Novel Non-Lanthanide Contrast Agent for X-ray Computed Tomography and T1-Weighted Magnetic Resonance Imaging. Nanoscale 2014, 6, 22112217. (24) Liu, Z.; Liu, X.-J.; Ran, X.; Ju, E.-G.; Ren, J.-S.; Qu, X.-G. Single-Layer Tungsten Oxide as Intelligent Photo-Responsive Nanoagents for Permanent Male Sterilization. Biomaterials 2015, 69, 56-64. (25) Liu, Z.; Liu, J.-H.; Wang, R.; Du, Y.-D.; Ren, J.S.; Qu, X.-G. An Efficient Nano-Based Theranostic System for Multi-Modal Imaging-Guided Photothermal Sterilization in Gastrointestinal Tract. Biomaterials 2015, 56, 206-218. (26) Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of Nanomaterials. Chem. Soc. Rev. 2012, 41, 2323-2343.

14

ACS Paragon Plus Environment

Page 15 of 16

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

ACS Applied Materials & Interfaces

(27) Nishiyama, N.; Stapert, H. R.; Zhang, G.-D.; Takasu, D.; Jiang, D.-L.; Nagano, T.; Aida, T.; Kataoka, K. Light-Harvesting Ionic Dendrimer Porphyrins as New Photosensitizers for Photodynamic Therapy. Bioconjugate Chem. 2003, 14, 58-66. (28) Zhang, D.; Wu, M.; Zeng, Y.-Y.; Wu, L.-J.; Wang, Q.-T.; Xiao Han, X.; Liu, X.-L.; Liu, J.-F. Chlorin e6 Conjugated Poly(dopamine) Nanospheres as PDT/PTTDual-Modal Therapeutic Agents for Enhanced Cancer Therapy. ACS Appl. Mater. Interfaces 2015, 7, 8176-8187. (29) Mahmood, Z.; Xu, K.-J.; Küçüköz, B.; Cui, X.N.; Zhao, J.-Z.; Wang, Z.-J.; Karatay, A.; Yaglioglu, H. G.; Hayvali, M.; Elmali, A. DiiodoBodipyPerylenebisimide Dyad/Triad: Preparation and Study of the Intramolecular and Intermolecular Electron/Energy Transfer. J. Org. Chem. 2015, 80, 3036-3049. (30) Wang, Z.-Z.; Chen, Z.-W.; Liu, Z.; Shi, P.; Dong, K.; Ju, E.-G.; Ren, J.-S.; Qu, X.-G. A Multi-Stimuli Responsive Gold Nanocageehyaluronic Platform for Targeted Photothermal and Chemotherapy. Biomaterials 2014, 35, 9678-9688. (31) Mu, J.-N.; Meng, X.-Y.; Chen, L.; Lu, Z.-B.; Mou, Q.-W.; Li, X.-X.; Wang S.-Q.; Yue, H.-Y. Highly stable and biocompatible W18O49@PEG-PCL hybrid nanospheres combining CT imaging and cancer photothermal therapy. RSC Adv. 2017, 7, 10692-10699. (32) Maeda, H.; Tsukigawa, K.; Fang, J. A Retrospective 30 Years After Discovery of the Enhanced Permeability and Retention Effect of Solid Tumors: Next‐ Generation Chemotherapeutics and Photodynamic Therapy—Problems, Solutions, and Prospects. Microcirculation 2016, 23, 173-182. (33) Fang, J.; Long, L.; Maeda, H. Enhancement of Tumor-Targeted Delivery of Bacteria with Nitroglycerin Involving Augmentation of the EPR Effect. Methods Mol. Biol. 2016, 1409, 9-23. (34) Maeda, H. Toward a Full Understanding of the EPR Effect in Primary and Metastatic Tumors as Well as Issues Related to Its Heterogeneity. Adv. Drug Delivery Rev. 2015, 91, 3-6.

15

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 16 of 16

Table of Contents

16

ACS Paragon Plus Environment