Superstable Magnetic Nanoparticles in Conjugation with Near-Infrared

Subscriber access provided by GAZI UNIV

Article

Superstable Magnetic Nanoparticles in Conjugation with Near-infrared Dye as a Multimodal Theranostic Platform Huige Zhou, Xiaoyang Hou, Ying Liu, Tianming Zhao, Qiuyu Shang, Jinglong Tang, Jing Liu, Yuqing Wang, Qiuchi Wu, Zehao Luo, Hui Wang, and Chunying Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11308 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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 21

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

Superstable Magnetic Nanoparticles in Conjugation with Near-infrared Dye as a Multimodal Theranostic Platform Huige Zhoua, Xiaoyang Houa, Ying Liua,*, Tianming Zhaoa,b, Qiuyu Shanga,b, Jinglong Tanga, Jing Liua, Yuqing Wanga, Qiuchi Wua,b, Zehao Luoa,b, Hui Wanga,b and Chunying Chen a,c,* a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for

Nanoscience and Technology of China, Beijing 100190, P.R. China b

School of Material Science and Engineering, University of Science and Technology Beijing, Beijing

100083, P.R. China c

School for Radiological and Interdisciplinary Sciences (RAD-X) & Collaborative Innovation Center of

Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215000, China

*Corresponding authors. Tel: +86 10 82545560; fax: +86 10 62656765 E-mail address: [email protected]

(C. Chen); [email protected] (Y. Liu)

ACS Paragon Plus Environment


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

ABSTRACT: Near-infrared (NIR) dyes functionalized magnetic nanoparticles (MNPs) have been widely applied in magnetic resonance imaging (MRI), NIR Fluorescence imaging, drug delivery and magnetic hyperthermia. However, the stability of MNPs and NIR dyes in water is a key problem to be solved for long-term application. In this study, a kind of superstable iron oxide nanoparticles was synthesized by a facile way, which can be used as T1 and T2 weighted MRI contrast agent. IR820 was grafted onto the surface of nanoparticles by 6-amino hexanoic acid to form IR820-CSQ-Fe conjugates. Attached IR820 showed increased stability in water at least for three month and enhanced ability of singlet oxygen production of almost double of free dyes, which will improve its efficiency for photodynamic therapy. Meanwhile, the multispectral optoacoustic tomography (MSOT) and NIR imaging ability of IR820-CSQ-Fe will greatly increase the accuracy of disease detection. All of these features will broaden the application of this material as a multimodal theranostic platform. KEYWORDS: multimodal imaging; stability; NIR dyes; magnetic nanoparticles; enhanced photodynamic therapy;


Page 2 of 21

Page 3 of 21

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


INTRODUCTION Nanotheranostics combining therapy and diagnostic in one nanocarrier by nanobiotechnology is one of the attractive way to improve current cancer therapies in complicated in vivo physiological environment.1, 2 Integration of imaging capability into drug delivery system will make it possible to monitor the real time distribution of nanocarriers.3 Magnetic resonance imaging, multispectral optoacoustic tomography (MSOT) imaging and near infrared imaging attract the most interest. Magnetic resonance imaging (MRI) provide harmless, excellent deep tissue contrast and spatial resolution, but the disadvantages are that it is a nonquantitative and time consuming method which may produce movement artifact.4 Optical imaging, by contrast, is time saving and sensitive. It provides excellent spatial and temporal resolution. However, the penetration of this method is limited to a few millimeters beneath the skin surface, so do the near-infrared (NIR) dyes.5 Although multispectral optoacoustic tomography (MSOT) imaging is better by combining good spectral selectivity of laser light and high resolution of ultrasound detection, it is still not enough to detect deeper tissues.6, 7, 8 All of the above mentioned methods have their superior advantages while each of them has some limits and is not sufficient enough to obtain all the necessary information.9 Thus, the development of one contrast agents which can validate various kinds of imaging with low toxicity is very urgent for precision diagnosis. One of the wonderful approaches is to combine magnetic nanoparticles with NIR dyes.10, 11 New Indocyanine Green (IR820) is a NIR organic dye providing unique absorption bands in wavelength range 600-1000 nm, and can be used as photodynamic therapy (PDT) agents.12 Compared with Indocyanine green (ICG), which has been approved by the Food and Drug Administration (FDA), IR820 own the meso-halogen in the center makes further conjugation available.13 Although the chemical structure changes make IR820 more stable under water and lighting condition, the singlet oxygen production of IR820 decreasing to half of that of ICG at the same concentration, which will reduce the therapeutic efficiency of PDT. In addition, the stability of IR820 is not enough for long-time observation. With good biocompatibility, unique magnetic properties and comparable size, iron oxide nanoparticles have shown great promise for biological diagnostic and therapeutic applications.14 Especially for superparamagnetic nanoparticles with small diameters, they exhibit a tremendous potential to be used as T1 and T2 weighted MRI contrast agents.15 Compared with other MRI contrast agents, which own their T1 and T2 weighted imaging ability by combining gadolinium or Manganese elements with iron oxide16, 17, 18, iron oxide alone is much safer. However, ligand exchange is necessary since the stability of magnetic nanoparticles is not good in water by one step of thermal decomposition, and this will make the synthesis method complicated.17 Thus, another method was adopted to synthesis water-soluble magnetic nanoparticles by coprecipitation of iron ion in alkaline polymer aqueous solution.15, 19 Chitosan quaternary ammonium salt (CSQ), which is biocompatibility and often used as an antimicrobial agent, was applied to synthesize MRI contrast agent.20, 21, 22, 23 There are two main methods to formulate magnetic nanoparticles (MNPs) for



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

combining MRI and optical imaging, one is encapsulation of iron-oxide and fluorophores in emulsions or polymeric nanocapsules24, 25, 26, and the other is conjugating dyes to the surface of nanoparticles.27, 28, 29 The second way is superior to the first one in the stability of nanoparticles in physiological environment. In this study, we synthesized a novel theranostic agent, ferroferric oxide nanoparticles capped with IR820 grafted onto chitosan quaternary ammonium salt (IR820-CSQ-Fe), as shown in Scheme 1. IR820-CSQ-Fe nanoparticles are favorable to be used as a multimode contrast agent and photodynamic therapy agent. Surprisingly, we found that grafted IR820 onto the surface of CSQ-Fe can increase its stability in water for at least three months and improve the PDT efficiency almost double than free IR820.

Scheme 1 Schematic illustration of the formation of IR820-CSQ-Fe NPs for combined MRI/MSOT/FI imaging and PDT.

EXPERIMENTAL SECTION


Page 4 of 21

Page 5 of 21

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


Materials All solvents and reagents were purchased from Beijing Chemical Works if not stated otherwise. Chitosan quaternary ammonium salt (Chitosan-N-2-hydroxy-N,N,Ntrimethylpropan-1-amonium chloride (HACC) CSQ, Mw = 100 kDa) was purchased from the Dongying Tianhua biological additives Co. Ltd. (Shandong, China). IR820 was purchased from Suzhou BEC Biological Technology Co., Ltd. (Beijing, China). 6-Aminocaproic acid was purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and triethylamine (TEA) were purchased from AlfaAesar (Ward Hill, MA, USA). Singlet Oxygen Sensor Green (SOSG) was purchased from life technology. Characterization TEM images were conducted with FEI Tecnai G2 microscope at an accelerating voltage of 200KV. The absolute concentrations of Fe in each sample were measured by ICP-OES (PE8000, Perkin Elmer, USA). Dynamic light scattering (DLS) measurements were carried out by a Zeta Sizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). For Fourier-transform infrared spectroscopy (FT-IR) measurements, potassium bromide (KBr) and materials were mixed in a mass ratio of 1:50 to form transparent tablets, and were detected with Fourier-transform infrared spectroscopy (FT-IR). The relaxation measurements and material/cell magnetic resonance images were performed on a 7.0T small animal MRI instrument (BioSpec70/20USR)，Bruker. Sample absorption spectrum was measured from 400– 900 nm in 5-nm intervals with a Cary UV spectrophotometer (Perkin Elmer Lambda 850). Sample emission spectra were recorded from 690 nm to 850 nm after excitation at 655 nm using a Fluorolog-3 spectrofluorometer (F-4600，HITACHI，Japan). Synthesis of CSQ-Fe Conjugate CSQ-Fe (CSQ-Fe3O4) was synthesized through high temperature coprecipitation. 19 Briefly, 2g CSQ was dissolved in 50mL deionized water bubbled with nitrogen for 30min. Then, the solution was heated up to 102 oC by an oil bath. At the same time, FeCl3∙6H2O (0.1459 g, 0.54 mmol) and FeCl2∙4H2O (0.0715 g, 0.36 mmol) were dissolved in diluted HCl solution (2 mL, 1 M). After that, the mixture of iron precursors was quickly injected into the hot polymer solution in a nitrogen atmosphere with vigorous stirring, followed by drop addition of ammonia water (15 mL, 28 %) to adjust the pH value to 9-10. The reaction system was cooled to room temperature after refluxing for 40 min and dialyzed for 72 h in a Spectra/Por, (MWCO: 12000-14000) dialysis bag against deionized water to remove unreacted reagents. Then the colloid in the dialysis bag was filtered by a 0.45 µm filter membrane and collected in a glass bottle. Synthesis of IR820-CSQ-Fe Nanoparticles Taking some of the CSQ-Fe colloide and lyophilized to powder. IR820 was grafted onto CSQ-Fe as describe before but with minor modifications.13 Briefly, it was



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

divided into two steps. The first step was to prepare IR-linker, involved substitution of the meso-chlorine group of IR820 with a linker 6-amino hexanoic acid to form a carboxyl-derivatized dye (IR-linker, Solution1). IR820 dye (15 mg, 0.0177 mmol) was reacted with 6-aminohexanoic acid (16.25 mg, 0.1239 mmol) in 2 ml anhydrous N′, N′-Dimethyl Formamide with magnetic stirring. Nitrogen gas was purged into the solution for 30 min, and then, triethylamine (17 μL, 0.1239 mmol) was injected into the reaction solution. The resulting green solution was stirred for 3 h in a three-necked, round-bottomed flask submerged into an oil bath to maintain a constant temperature of 85 oC during the procedure. The color of the solution turned blue when the reaction is completed. The IR-linker provided suitable functional groups as well as paved the way for less hindered substitution of polymers. In the second step, the IR820-CSQ-Fe conjugate was prepared by activating the carboxyl-derivatized dye (IR-linker) with EDC and NHS, in order to allow conjugation with the imino group of CSQ. Briefly, an equimolar solution of EDC, NHS and IR-linker was allowed to react in 9 ml anhydrous DMF. This step helped the IR-linker to form an ester intermediate making the next reaction easy. Next, 5 ml of CSQ-Fe colloid (contained 77.8 mg CSQ-Fe) was added to the solution dropwise. After reacting overnight at room temperature, the mixture was dialyzed for 48h in a Spectra/Por, (MWCO: 12000-14000) dialysis bag against deionized water to remove excess unreacted agents and the final product (IR820-CSQ-Fe) was lyophilized. Synthesis of FITC Labeled CSQ-Fe FITC labeled CSQ-Fe was synthesized according to the reference with a few modifications. Briefly, 15 mg of CSQ-Fe was dissolved in 1 mL of H2O, and then 0.3 mL dimethyl sulfoxide solution of FITC (1mg/L) was added. The mixture was reacted at room temperature for 2 h in darkness. After reaction, the solution was then dialyzed against distilled water for 3 d and lyophilized subsequently. MSOT Signals A model MSOT scanner was utilized in this study (MSOT inVision 128, iThera medical, Germany). IR820-CSQ-Fe aqueous with different IR820 concentrations were embedded in agarose gel as the in vitro model to test the signal intensity. Cell Culture Human breast cancer cell lines MDA-MB-231 and human bronchial epithelial cell lines 16HBE(American Type Culture Collection, Rockville, Maryland, USA) were kept in the laboratory. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (Gibco), 2 mM L-glutamine, 20 mM HEPES, 100 U/mL penicillin and 1 µg/mL streptomycin (Invitrogen), in a humidified atmosphere of 5 % CO2 at 37 °C. In Vitro Cytotoxicity Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was applied to measure the cell viability after IR820-CSQ-Fe, CSQ-Fe and IR820 exposure, respectively. Briefly,


Page 6 of 21

Page 7 of 21

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


MDA-MB-231 cells or 16HBE cells were seeded in 96 well plates, and treated with IR820, CSQ-Fe and IR820-CSQ-Fe in different concentrations for 24 h. After exposure, 100 μL media containing CCK-8 (10%) was added and incubate at 37 °C for 2 h, the absorbance at 450 nm was measured using a multifunctional microplate reader (Infinite 200, TECAN). Cell viability of untreated MDA-MB-231 cells was considered as 100%. Each sample was triplicated and data represented the mean value of all measurements. In Vitro MR imaging MDA-MB-231 cells were seeded in 10 cm culture dishes, and exposed with IR820-CSQ-Fe with [Fe] concentration 0, 0.1 and 0.5 µM for 2 h. After exposure, cells were washed three times with PBS and then trypsinized and centrifuged to the bottom of 0.5 mL tube for MR imaging. NIR Imaging of IR820-CSQ-Fe The ability of as-synthesized nanoparticles to be used as a NIR fluorescence agent and the cellular uptake were examined using confocal laser scanning microscopy (CLSM) (Olympus FV1000). Briefly, MDA-MB-231 cells were exposed to 4 µg/mL IR820 concentration of IR820-CSQ-Fe ([Fe] =8.2 µg/mL) or FITC-CSQ-Fe ([Fe]=8.2 µg/mL) for 5 min, 30 min and 3 h, respectively. After exposure, cells were washed three times with PBS and fixed with 4 % paraformaldehyde for 15 min. Cells were then washed three times with PBS and stained with Hoechst 33342 (invitrogen) for 15 min and Lyso-Tracker Red (invitrogen) for 35 min at room temperature. Exicitation/Emission for Hoechst 33342, Lyso-Tracker Red, IR820-CSQ-Fe and FITC are 350/460, 577/590, 655/755 and 488/520. Cellular Uptake of IR820-CSQ-Fe and CSQ-Fe by MDA-MB-231 Cells at Different Incubation Time or Different Concentrations MDA-MB-231 cells seeded in 6-well plates (1×105 cells well-1) were treated with FITC-CSQ-Fe at different concentrations ([Fe]=2.5, 5, 10 and 20 μg mL-1) for 3 h at 37 °C and FITC-CSQ-Fe ([Fe]=10 μg mL-1) for 0, 1, 3 and 6h. At the end of incubation, cells were trypsinized, resuspended in the medium, washed with PBS thrice and then, resuspended in 0.5 mL PBS. The intracellular fluorescence was determined by FACSCalibur system. Measurement of Increased Temperature and Singlet Oxygen Generation NIR laser (808 nm, 8W/cm2) irradiated the aqueous of IR820 (4 µg/mL) and IR820CSQ-Fe (contain 4 µg/mL IR820) for 7 min. The temperature of aqueous at different time points was recorded by a thermal imager. SOSG were used to detect the production of singlet oxygen. Mixed SOSG (0.5 mL, 4 µg/mL) and IR820-CSQ-Fe (0.5 mL, 4 µg/mL IR820), and then NIR laser (808 nm, 8 W/cm2) irradiate the mixture for 0.5 min, 1 min, 2 min, 3 min. The signal intensity was recorded at 531 nm after excitation at 507 nm. Each test was triplicated.



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

Photodynamic tTerapy in Vitro MDA-MB-231 cell were seeded in 24-well plate at a density of 1×104 cells per well. Different IR820 concentrations of IR820-CSQ-Fe were added to expose cells for 2 h. After exposure, the cells were irradiated with a laser diode at 808 nm wavelength with 8 W/cm2 light density for 5 min. Then, calcein acetoxymethyl ester (calcein AM) (2µg/ml) and propidium iodide (PI) (2µg/ml) were used to stain live and dead cells to evaluate the photothermal effect of the nanoparticles on cancer cells. Picture of live and dead cells were taken under a fluorescence microscope (Olympus IX71, Japan).

RESULTS AND DISCCUSSION Synthesis and Characterization of IR820-CSQ-Fe Nanoparticles IR820-CSQ-Fe nanoparticles were synthesized by two steps. The first step was the preparation of CSQ-Fe by high temperature coprecipitation, and the second step was grafting IR820 onto the surface of CSQ-Fe with 6-aminocaproic acid as a macromolecule linker. Zeta potential of CSQ-Fe (Fig. S1) represented CSQ was successfully coated on the surface of Fe3O4 and the energy dispersive X-ray (EDX) spectrum (Fig. 1B) confirms that CSQ-Fe contained the elements of O and Fe. The TEM images (Fig. 1A) shows the uniformity of CSQ-Fe and the average size of the core of CSQ-Fe is 11.9 nm (Fig. S6). Compared with CSQ-Fe, the FT-IR spectrum of IR820-CSQ-Fe at 1653-1476 cm-1 changed because of the double benzene nucleus of IR820 and the existence of N-H in amide bond between IR-linker and CSQ-Fe. Zeta potential of IR820-CSQ-Fe had a slight left shift because of the sulfonic groups of IR820 (Fig. S2). Fig. 1D shows that the UV–visible spectrum of IR820-CSQ-Fe has peak absorption at 655 nm, whereas CSQ-Fe didn’t show obvious peak in 500-900 nm and the free dyes showed peak absorption at 690 nm. This result is in accordance with the previous report and verifies the successful graft of IR820.13 The distribution of hydrodynamic size of CSQ-Fe and IR820-CSQ-Fe can be observed from Fig. 1E, and this result showed that grafted IR820 didn’t affect the morphology of CSQ-Fe. The element content of Fe for each sample was detected by ICP-OES analysis and the dye content of IR820-CSQ-Fe was detected by the light absorbance of 688 nm. There is 0.035 mg Fe and 0.072 mg IR820 in per milligram materials. The percentage of IR820 substitution in CSQ was approximately 3.01 %, which is much higher than that reported by Srinivasan 30.


Page 8 of 21

Page 9 of 21

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


Fig. 1 Characterization of CSQ-Fe and IR820-CSQ-Fe; (A) TEM images and (B) EDX of CSQ-Fe; (C) FT-IR spectrum of CSQ-Fe and IR820-CSQ-Fe; (D) UV-vis absorbance of IR820 ([IR820]=47 µg/mL), CSQ-Fe and IR820-CSQ-Fe ([IR820]= 49µg/mL) in water; (E) Hydrodynamic size distribution of CSQ-Fe and IR820-CSQ-Fe; (F) Fluorescence spectrum of IR820-CSQ-Fe (Ex: 655 nm, [IR820]=12 µg/mL). Long-time Stability of IR820-CSQ-Fe in Water The UV–visible light absorbance stability of dyes in water is a very important property for its biological applications especially for long time observation. Compared with free IR820, the light absorbance intensity of IR820-CSQ-Fe slightly decreased in the absorption peak (from 1.56 to 1.32), whereas IR820 had a sharp



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

reduction (from 1.51 to 0.99) (Fig. 2A and 2B). After three months exposure at room temperature in darkness, the color of IR820 aqueous solution was bleached while IR820-CSQ-Fe colloid solution had negligible changes and showed no precipitate, as shown in Fig. 2C. Conjugation to CSQ-Fe nanoparticles makes IR820 more stable in water and these features will enlarge the application for long-term survey. The high colloid stability may due to the positive charge on the surface of CSQ-Fe and IR820-CSQ-Fe (Fig. S1 and Fig. S2).

Fig. 2 Stability analysis of IR820-CSQ-Fe; UV-vis absorbance spectrum of (A) IR820 ([IR820]=47 µg/mL) and (B) IR820-CSQ-Fe ([IR820]=49 µg/mL) different time at day 1, 2 and 8. (C) Photos of IR820, CSQ-Fe and IR820-CSQ -Fe after store at room temperature in darkness for one day and three month. Cell Viability Assessment Biological safety is a very important feature of materials used as a theranostic agent. In order to test the safety of CSQ-Fe and IR820-CSQ-Fe in cell level, different concentrations of CSQ-Fe and IR820-CSQ-Fe nanoparticles were added into the culture medium of MDA-MB-231 cells or 16HBE cells and incubated for 24 h. Fig. 3A shows that CSQ-Fe exposure had negligibly influence on cell viability up to 80 µg Fe /mL, which suggests it could be a safe MRI contrast agent. As shown in Fig. 3B & 3D, cell viability of MDA-MB-231 cells and 16HBE cells incubated with IR820-CSQ-Fe had a decrease 16 % and 14% at IR820 concentrations up to 4 µg/mL,


Page 10 of 21

Page 11 of 21

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


respectively. The protonation of CSQ residues could be attributed to the slight toxicity of IR820-CSQ-Fe, which increases the interactions between this polycationic polymer and cell membranes.

Fig. 3 Viability of MDA-MB-231 cells after different treatments: (A) CSQ-Fe, (B) IR820-CSQ-Fe, (C) IR820, and (D) Viability of 16 HBE cells after treatments with free IR820 and IR820-CSQ-Fe. Linear Relationship of MSOT Signals vs the Concentration of IR820-CSQ-Fe The MSOT signal of IR820-CSQ-Fe has a good linear relationship with the dye concentration, as shown in Fig. 4A. Different dye concentration of IR820-CSQ-Fe has the different signal intensity for different brightness (Fig. 4B). This result validates the ability of these nanoparticles to be used as a MSOT imaging contrast agent to observe therapy effect or guide drug delivery.



A Signal Intensity (a.u.)

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

8000

6000

y=2661x-176 R2=0.989

4000

2000

1

2

3

Concentration (g/mL)

B

Fig. 4 MSOT signal of IR820-CSQ-Fe; (A) Plot of MSOT signal intensity of IR820-CSQ-Fe at different IR820 concentrations; (B) MSOT images of IR820-CSQ-Fe at different IR820 concentrations. Enhanced Relaxometric Properties of IR820-CSQ-Fe and T1&T2 MR Imaging Fig 5 A and B shows the T1 and T2 relaxivity of IR820-CSQ-Fe. Both of the two relaxations are enhanced as the concentration of Fe increases, resulting in a positive T1 contrast and a negative T2 contrast. T1&T2 MR imagings of materials with different Fe concentration were shown in Fig 5 C. As expected, T1 weighted imagings become brighter as Fe concentration increased, while T2 weighted imagings become darker. In another interesting work29, the specific relaxivity (r2) was estimated to be 4.41 mM-1s-1 and without T1 relaxivity. Our materials showed a significant enhancement of T2 relaxivity with the much high r2 value (440.6 mM-1s-1) and owned the T1 relaxivity imaging affect (r1=1.180 mM-1s-1). In order to test the T1 and T2 MRI images in vitro, MDA-MB-231 cells were used to incubate with IR820-CSQ-Fe at various Fe concentrations (0, 0.1, 0.5 µM). As shown in Fig. 5D, compared with cells without IR820-CSQ-Fe exposure, the cells treated with IR820-CSQ-Fe were brightened on the top and darkened in the bottom (on the left of Fig. 5D). Combined with the color images (on the right of Fig. 5D), we know that both of T1 and T2 signal intensities are gradually enhanced as the concentration of Fe increased. From the concentration ratio of IR820 and Fe within IR820-CSQ-Fe, we can know that Fe concentration of IR820-CSQ-Fe is 34 µM when IR820 concentration is 4 µg/mL which is much higher than the dose of it used in cell imaging. These results indicated that IR820-CSQ-Fe has excellent MRI contrast effect at low dose that will reduce the side effect of to patients.


Page 12 of 21

Page 13 of 21

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


A

C

B

D

Fig. 5 Relaxometric properties and MR imaging; (A) Plot of R 1 versus Fe concentration; (B) Plot of R2 versus Fe concentration; (C) T1 and T2 weighted MR images of aqueous solutions containing different Fe concentration of IR820-CSQ-Fe; (D) T1 and T2 weighted MR images of MDA-MB-231 cells labeled by IR820-CSQ-Fe.

NIR Imaging of IR820-CSQ-Fe for Cellular Uptake NIR dyes with a Large Stokes shift are very promising in application of biological imaging31. As synthesized IR820-CSQ-Fe absorbs at 655 nm (Fig. 1F) and emits at 804 nm (Fig. 2B) displaying a larger Stokes shift (149 nm) than that of free IR820 (130 nm)12. This feature will makes it more suitable for biological optical imaging. Figure 6A shows an array of cells stained with Hoechst 33342 (blue), lysotracker (red), IR820-CSQ-Fe (green), and the merged images at 5 min, 30 min and 3 h of incubation with IR820-CSQ-Fe nanoparticles. The green fluorescence observed in MDA-MB-231 cells indicated that IR820-CSQ-Fe nanoparticles were taken up by cells. The yellow color merged images indicate the co-localization of IR820-CSQ-Fe nanoparticles with intercellular lysosomes. Compared with images of 5 min, the yellow color is increasing indicated that more nanoparticles were internalized after 30 min. However, the green color is increased at 3 h. Once entered into cells, the protonation of numerous amino groups on the surface of IR820-CSQ-Fe makes the endosomal membrane destabilize so that them could escape from lysosome, which is often called “proton sponge” effect.13 This result confirms that IR820-CSQ-Fe nanoparticles are easy to be internalized by cells and the NIR fluorescence property of



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

IR820-CSQ-Fe is favorite for imaging. Compared with IR820-CSQ-Fe, FITC-CSQ-Fe nanoparticles have almost the same hydrodynamic size distribution (Fig. S6) and zeta potential (Fig. S7). Besides, their cellular uptake behaviors are quite similar (Fig. S3). Therefore, we could use FITC-CSQ-Fe to quantify the uptake of IR820-CSQ-Fe by cells through flow cytometry. Fig. 6B shows that the fluorescence intensity of cells increased with the concentration increasing at the same incubation time and it also increased with the incubation time extending at the same concentration. These results show that cellular uptake of IR820-CSQ-Fe is quick and has good time- and concentration- dependent profiles. A

B1

B2


Page 14 of 21

Page 15 of 21

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


Fig. 6 Cellular uptake of IR820-CSQ-Fe; (A) CLSM images of MDA-MB-231 cells after incubation with IR820-CSQ-Fe (4µg/mL IR820-CSQ-Fe) for 5min, 30 min and 3 hours. The blue fluorescence of nucleus, the red fluorescence of lysosomal and the green fluorescence IR820 of IR820-CSQ-Fe were analyzed by a confocal microscope (scale = 20μm). (B) Amounts of FITC-CSQ-Fe uptaken in MDA-MB-231 cells after different incubation deals determined by Flow cytometric: concentration dependence (B1) and time dependence (B2). Improved Generation of Singlet Oxygen by IR820-CSQ-Fe for Photodynamic Therapy Thermal therapies, such as photothermal therapy32-34 and magnetic fluid hyperthermia35-37, need high temperature to kill the cancer cells. In contrast, the mechanism of PDT killing cancer cells is involved in producing highly reactive oxygen species, especially singlet oxygen (1O2).38, 39 The production of singlet oxygen of IR820-CSQ-Fe increased almost double of free IR820 at the same IR820 concentration, which will absolutely increase the efficiency to kill cancer cells. Compared with ICG, the chemical structure of IR820 is more stable under different temperature and lighting conditions. The bridge between indole rings makes degradation half-times for IR820 approximately doubled than ICG.40 However, IR820 produce less singlet oxygen than ICG at the same concentration, which can be observed from Fig 7B. Whereas, attach IR820 on the surface of CSQ-Fe could increase the singlet oxygen production of IR820 to the same level of ICG. From Fig. 7B, the singlet oxygen generation of CSQ-Fe has not increased obvious compared with free and conjugated IR820. However, from the absorption spectrum of IR820-CSQ-Fe, we can know that the improved singlet oxygen production is probably due to the interaction of long polymer chains on the surface of IR820-CSQ-Fe with IR820 and among IR820 form both H and J aggregation just like previous reports29, 41. Although the temperature of aqueous solution of IR820-CSQ-Fe nanoparticles will increase under laser irradiation (Fig. S4), the rise of temperature is not obvious at low concentration (Fig. 7A). Therefore, from Fig. 7C-H, we know that the death of MDA-MB-231 cells is caused of singlet oxygen rather than high temperature. After irradiated with 808nm laser at 8 W cm-2, free IR820 and grafted IR820 have similar temperature increase. However, IR820-CSQ-Fe with lower IR820 concentration could not generate enough singlet oxygen to kill cells (Fig. 7E and 7F, 25.3% and 30.9%). When IR820 concentration of IR820-CSQ-Fe increased to 4 µg/mL, most of cells died (66.5%), indicated as red cells. Although free IR820 could heat up media to the same temperature as IR820-CSQ-Fe, they could not product adequate singlet oxygen to damage the plenty of cells at the same dye concentration as IR820-CSQ-Fe (Fig. 7H).



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

B

A

C

D

E

F

G

H

I

Fig. 7 Generation of singlet oxygen and photodynamic therapy in vitro; (A) Temperature increase of water, IR820 and IR820-CSQ-Fe; (B) Plot of the intensity of the singlet oxygen signal over irradiation time; (C) Control cells without exposure and irradiation; (D) Cells with 4 µg/mL IR820-CSQ-Fe exposure, but without irradiation; (E) Cells with 0.8 µg/mL IR820-CSQ- Fe exposure and 5 min irradiation; (F) Cells with 2 µg/mL IR820-CSQ- Fe exposure and 5 min irradiation; (G) Cells with 4 µg/mL IR820-CSQ- Fe exposure and 5 min irradiation; (H) Cells with 4 µg/mL IR820 exposure and 5 min irradiation; (I) Statistical data of percentage of cell viability and the abscissa according to Fig 7C-H.


Page 16 of 21

Page 17 of 21

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


CONCLUSIONS We have successfully designed and validated a novel multimodal theranostic agent by grafting IR820 onto the surface of magnetic CSQ-Fe for combined MRI/MSOT/FI imaging and photodynamic therapy. The synthesis method for IR820-CSQ-Fe is simple and facile to manipulate. More importantly, the IR820-CSQ-Fe nanosystem can doubly increase the production of singlet oxygen than free IR820 dyes and make IR820 stable in water for at least three months which makes the long-term observation available. In addition, the spectral blue shift of modified IR820 will broaden the application as a NIR fluorescence imaging agent. MSOT signal test and MRI imaging test also prove that as synthesized IR820-CSQ-Fe has the ability to be used as T1/T2 MRI and MSOT contrast agent, which provides a facile platform for cancer treatment and detection.

ASSOCIATED CONTENT Supporting Information Zeta potential data, cellular uptake of FITC-CSQ-Fe, photothermal effects data, fluorescence spectrum of IR820, TEM data, hydrodynamic size distribution of FITC-CSQ-Fe, photos of nanomaterials in PBS with different pH and DMEM, 1H NMR spectrum of IR820-CSQ-Fe. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail address: [email protected]. Tel: +86 10 82545560; fax: +86 10 62656765. E-mail address: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology of China (National Basic Research Programs 2012CB934000), the International Science & Technology Cooperation Program of China, the Ministry of Science Technology of China (2014DFG52500), the National Science Foundation of China (21320102003, 21403043) and the National Science Fund for Distinguished Young Scholars (11425520).

REFERENCES (1) Melancon, M. P.; Stafford, R. J.; Li, C. Challenges to Effective Cancer Nanotheranostics. J. Controled Release 2012, 164, 177-182.



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

(2)

Kievit, F. M.; Zhang, M. Cancer Nanotheranostics: Improving Imaging and Therapy by

Targeted Delivery Across Biological Barriers. Adv. Mater. 2011, 23, H217-H247. (3)

Wu, M.; Meng, Q.; Chen, Y.; Xu, P.; Zhang, S.; Li, Y.; Zhang, L.; Wang, M.; Yao, H.; Shi, J.

Ultrasmall Confined Iron Oxide Nanoparticle MSNs as a pH-Responsive Theranostic Platform. Adv. Funct. Mater. 2014, 24, 4273-4283. (4)

Baker, M. Whole-Animal Imaging: The Whole Picture. Nature 2010, 463, 977-980.

(5)

Weissleder, R.; Pittet, M. J. Imaging in the Era of Molecular Oncology. Nature 2008, 452,

580-589. (6)

Ntziachristos, V.; Razansky, D. Molecular Imaging by Means of Multispectral Optoacoustic

Tomography (MSOT). Chem. Rev. 2010, 110, 2783-2794. (7)

Li, J. Nanotechnology-based Platform for Early Diagnosis of Cancer. Sci. Bull. 2015, 60,

488-490. (8)

Fan, Q.; Cheng, K.; Hu, X.; Ma, X.; Zhang, R.; Yang, M.; Lu, X.; Xing, L.; Huang, W.;

Gambhir, S. S.; Cheng, Z. Transferring Biomarker into Molecular Probe: Melanin Nanoparticle as a Naturally Active Platform for Multimodality Imaging. J. Am. Chem. Soc. 2014, 136, 15185-15194. (9)

Liu, J.; Zheng, X.; Yan, L.; Zhou, L.; Tian, G.; Yin, W.; Wang, L.; Liu, Y.; Hu, Z.; Gu, Z.;

Chen, C.; Zhao, Y. Bismuth Sulfide Nanorods as a Precision Nanomedicine for in Vivo Multimodal Imaging-Guided Photothermal Therapy of Tumor. ACS Nano 2015, 9, 696-707. (10)

Frimpong, R. A.; Hilt, J. Z. Magnetic Nanoparticles in Biomedicine: Synthesis,

Functionalization and Applications. Nanomedicine 2010, 5, 1401-1414. (11)

Zhang, W.; Gao, C. Recent Advances in Cell Imaging and Cytotoxicity of Intracellular

Stimuli-responsive Nanomaterials. Sci. Bull. 2015, 60, 1973-1979. (12)

Prajapati, S. I.; Martinez, C. O.; Bahadur, A. N.; Wu, I. Q.; Zheng, W.; Lechleiter, J. D.;

McManus, L. M.; Chisholm, G. B.; Michalek, J. E.; Shireman, P. K.; Keller, C. Near-Infrared Imaging of Injured Tissue in Living Subjects Using IR-820. Mol. Imaging 2009, 8, 45-54. (13)

Masotti, A.; Vicennati, P.; Boschi, F.; Calderan, L.; Sbarbati, A.; Ortaggi, G. A Novel

Near-Infrared Indocyanine Dye - Polyethylenimine Conjugate Allows DNA Delivery Imaging in Vivo. Bioconjugate Chem. 2008, 19, 983-987. (14)

Hao, R.; Xing, R. J.; Xu, Z. C.; Hou, Y. L.; Gao, S.; Sun, S. H. Synthesis, Functionalization,

and Biomedical Applications of Multifunctional Magnetic Nanoparticles. Adv. Mater. 2010, 22, 2729-2742. (15)

Li, Z.; Yi, P. W.; Sun, Q.; Lei, H.; Zhao, H. L.; Zhu, Z. H.; Smith, S. C.; Lan, M. B.; Lu, G.

Q. Ultrasmall Water-Soluble and Biocompatible Magnetic Iron Oxide Nanoparticles as Positive and Negative Dual Contrast Agents. Adv. Funct. Mater. 2012, 22, 2387-2393. (16)

Chen, J.; Zhang, W.-J.; Guo, Z.; Wang, H.-B.; Wang, D.-D.; Zhou, J.-J.; Chen, Q.-W.

Ph-Responsive Iron Manganese Silicate Nanoparticles as T1-T2* Dual-Modal Imaging Probes for Tumor Diagnosis. ACS Appl. Mater. Interfaces 2015, 7, 5373-5383. (17)

Im, G. H.; Kim, S. M.; Lee, D.-G.; Lee, W. J.; Lee, J. H.; Lee, I. S. Fe3O4/MnO Hybrid

Nanocrystals as a Dual Contrast Agent for Both T1- and T2-Weighted Liver MRI. Biomaterials. 2013, 34, 2069-2076. (18)

Zhou, Z.; Huang, D.; Bao, J.; Chen, Q.; Liu, G.; Chen, Z.; Chen, X.; Gao, J. A

Synergistically Enhanced T1-T2 Dual-Modal Contrast Agent. Adv. Mater. 2012, 24, 6223-6228. (19)

Wang, G.; Zhang, X.; Skallberg, A.; Liu, Y.; Hu, Z.; Mei, X.; Uvdal, K. One-Step Synthesis

of Water-Dispersible Ultra-Small Fe3O4 Nanoparticles as Contrast Agents for T1 and T2 Magnetic


Page 18 of 21

Page 19 of 21

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


Resonance Imaging. Nanoscale 2014, 6, 2953-2963. (20)

Tan, H.; Ma, R.; Lin, C.; Liu, Z.; Tang, T. Quaternized Chitosan as an Antimicrobial Agent:

Antimicrobial Activity, Mechanism of Action and Biomedical Applications in Orthopedics. Int. J. Mol. Sci. 2013, 14, 1854-1869. (21)

Gour, N.; Ngo, K. X.; Vebert-Nardin, C. Anti-Infectious Surfaces Achieved by Polymer

Modification. Macromol. Mater. Eng. 2014, 299, 648-668. (22)

Meng, H.; Wei, R. Use of Smart Designed Nanoparticles to Impact Cancer Surgery. Sci. Bull.

2015, 60, 142-143. (23)

Peng, Z.-X.; Wang, L.; Du, L.; Guo, S.-R.; Wang, X.-Q.; Tang, T.-T. Adjustment of the

Antibacterial Activity and Biocompatibility of Hydroxypropyltrimethyl Ammonium Chloride Chitosan by Varying the Degree of Substitution of Quaternary Ammonium. Carbohydr. Polym. 2010, 81, 275-283. (24)

Foy, S. P.; Manthe, R. L.; Foy, S. T.; Dimitrijevic, S.; Krishnamurthy, N.; Labhasetwar, V.

Optical Imaging and Magnetic Field Targeting of Magnetic Nanoparticles in Tumors. ACS Nano 2010, 4, 5217-5224. (25)

Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Drug/Dye-Loaded, Multifunctional Iron

Oxide Nanoparticles for Combined Targeted Cancer Therapy and Dual Optical/Magnetic Resonance Imaging. Small 2009, 5, 1862-1868. (26)

Qian, J.; Wang, D.; Cai, F.; Zhan, Q.; Wang, Y.; He, S. Photosensitizer Encapsulated

Organically Modified Silica Nanoparticles for Direct Two-Photon Photodynamic Therapy and in Vivo Functional Imaging. Biomaterials. 2012, 33, 4851-4860. (27)

Sun, C.; Du, K.; Fang, C.; Bhattarai, N.; Veiseh, O.; Kievit, F.; Stephen, Z.; Lee, D.;

Ellenbogen, R. G.; Ratner, B.; Zhang, M. PEG-Mediated Synthesis of Highly Dispersive Multifunctional Superparamagnetic Nanoparticles: Their Physicochemical Properties and Function in Vivo. ACS Nano 2010, 4, 2402-2410. (28)

Thierry, B.; Al-Ejeh, F.; Khatri, A.; Yuan, Z.; Russell, P. J.; Ping, S.; Brown, M. P.; Majewski,

P. Multifunctional Core-Shell Magnetic Cisplatin Nanocarriers. Chem. Commun. 2009, 7348-7350. (29)

Yen, S. K.; Janczewski, D.; Lakshmi, J. L.; Bin Dolmanan, S.; Tripathy, S.; Ho, V. H. B.;

Vijayaragavan, V.; Hariharan, A.; Padmanabhan, P.; Bhakoo, K. K.; Sudhaharan, T.; Ahmed, S.; Zhang, Y.; Selvan, S. T. Design and Synthesis of Polymer-Functionalized NIR Fluorescent Dyes-Magnetic Nanoparticles for Bioimaging. ACS Nano 2013, 7, 6796-6805. (30)

Srinivasan, S.; Manchanda, R.; Fernandez-Fernandez, A.; Lei, T.; McGoron, A. J.

Near-Infrared Fluorescing IR820-Chitosan Conjugate for Multifunctional Cancer Theranostic Applications. J. Photochem. Photobiol. B 2013, 119, 52-59. (31)

Peng, X. J.; Song, F. L.; Lu, E.; Wang, Y. N.; Zhou, W.; Fan, J. L.; Gao, Y. L. Heptamethine

Cyanine Dyes with a Large Stokes Shift and Strong Fluorescence: A Paradigm for Excited-State Intramolecular Charge Transfer. J. Am. Chem. Soc. 2005, 127, 4170-4171. (32)

Zhang, Z.; Wang, J.; Chen, C. Near-Infrared Light-Mediated Nanoplatforms for Cancer

Thermo-Chemotherapy and Optical Imaging. Adv. Mater. 2013, 25, 3869-3880. (33)

Zhang, Z.; Wang, J.; Nie, X.; Wen, T.; Ji, Y.; Wu, X.; Zhao, Y.; Chen, C. Near Infrared

Laser-Induced Targeted Cancer Therapy Using Thermoresponsive Polymer Encapsulated Gold Nanorods. J. Am. Chem. Soc. 2014, 136, 7317-7326. (34)

Zha, Z.; Yue, X.; Ren, Q.; Dai, Z. Uniform Polypyrrole Nanoparticles with High

Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells. Adv. Mater. 2013, 25,



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

777-782. (35)

Thomas, L. A.; Dekker, L.; Kallumadil, M.; Southern, P.; Wilson, M.; Nair, S. P.; Pankhurst,

Q. A.; Parkin, I. P. Carboxylic Acid-Stabilised Iron Oxide Nanoparticles for Use in Magnetic Hyperthermia. J. Mater. Chem. 2009, 19, 6529-6535. (36)

Jeun, M.; Bae, S.; Tomitaka, A.; Takemura, Y.; Park, K. H.; Paek, S. H.; Chung, K.-W.

Effects of Particle Dipole Interaction on the AC Magnetically Induced Heating Characteristics of Ferrite Nanoparticles for Hyperthermia. Appl. Phys. Lett. 2009, 95, 082501. (37)

Cao, M.J.; Wang, P.Y.; Kou, Y.; Wang, J.; Liu, J.; Li, Y.H.; Li, J.Y.; Wang, L.M.; Chen C.Y.

Gadolinium(III)-Chelated Silica Nanospheres Integrating Chemotherapy and Photothermal Therapy for Cancer Treatment and Magnetic Resonance Imaging. ACS Appl. Mater. Interfaces 2015, 7, 25014−25023. (38)

Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic Therapy and Anti-Tumour Immunity.

Nat. Rev. Cancer 2006, 6, 535-545. (39)

Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev.

Cancer 2003, 3, 380-387. (40)

Zhang, J. L.; Luo, W. M.; Zhu, Z. H. Photostability, Laser Energy Conversion Efficiency and

Absorption of IR Heptamethine Cyanine Dyes Absorbing Beyond 1µM. Dyes. Pigments 1995, 28, 1-6. (41)

Ornelas, C.; Lodescar, R.; Durandin, A.; Canary, J. W.; Pennell, R.; Liebes, L. F.; Weck, M.

Combining Aminocyanine Dyes with Polyamide Dendrons: A Promising Strategy for Imaging in the Near-Infrared Region. Chem-Eur. J. 2011, 17, 3619-3629.


Page 20 of 21

Page 21 of 21

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


TOC


Superstable Magnetic Nanoparticles in Conjugation with Near-Infrared

Recommend Documents