Journal name: International Journal of Nanomedicine Article Designation: Original Research Year: 2017 Volume: 12 International Journal of Nanomedicine Dovepress Running head verso: Yang et al Running head recto: HA-SPIONs for effective cancer diagnosis and treatment open access to scientific and medical research DOI: http://dx.doi.org/10.2147/IJN.S121249 Open Access Full Text Article ORIGINAL RESEARCH Hyaluronan-modified superparamagnetic iron oxide nanoparticles for bimodal breast cancer imaging and photothermal therapy Rui-Meng Yang1,* Abstract: Theranostic nanoparticles with both imaging and therapeutic abilities are highly Chao-Ping Fu2,* promising in successful diagnosis and treatment of the most devastating cancers. In this study, Jin-Zhi Fang1 the dual-modal imaging and photothermal effect of hyaluronan (HA)-modified superparamag- Xiang-Dong Xu1 netic iron oxide nanoparticles (HA-SPIONs), which was developed in a previous study, were Xin-Hua Wei1 investigated for CD44 HA receptor-overexpressing breast cancer in both in vitro and in vivo Wen-Jie Tang1 experiments. Heat is found to be rapidly generated by near-infrared laser range irradiation of HA- SPIONs. When incubated with CD44 HA receptor-overexpressing MDA-MB-231 cells in vitro, Xin-Qing Jiang1 HA-SPIONs exhibited significant specific cellular uptake and specific accumulation confirmed Li-Ming Zhang2 by Prussian blue staining. The in vitro and in vivo results of magnetic resonance imaging and 1Department of Radiology, Guangzhou photothermal ablation demonstrated that HA-SPIONs exhibited significant negative contrast First People’s Hospital, Guangzhou enhancement on T -weighted magnetic resonance imaging and photothermal effect targeted Medical University, 2School of 2 Materials Science and Engineering, CD44 HA receptor-overexpressing breast cancer. All these results indicated that HA-SPIONs School of Chemistry, Sun Yat-sen have great potential for effective diagnosis and treatment of cancer. University, Guangzhou, China Keywords: iron oxide nanoparticles, surface functionalization, bioactive glycosaminoglycan, *These authors contributed equally magnetic resonance imaging, cellular uptake, breast carcinoma to this work Introduction In recent years, near-infrared (NIR) laser-induced photothermal therapy (PTT) based on the nanotechnology, which can deliver a specific amount of photoenergy directly into the tumor site to increase treatment efficiency and meanwhile minimize the damage to the surrounding healthy tissues, has emerged as an appealing and minimally invasive therapeutic strategy for cancer treatment. Noble metal-based nanostructures, such as gold nanospheres,1,2 gold nanorods,3,4 gold nanoshells,5 gold nanocages,6 Cu-based semiconductor nanoparticles,7 and carbon nanotubes8 are the chief nanomaterials that have been well prepared and demonstrated powerful NIR laser-induced PTT efficacy. Correspondence: Xin-Qing Jiang Department of Radiology, Guangzhou However, their promising clinical applications were largely limited due to the innate First People’s Hospital, Guangzhou toxicity and unknown long-term toxicity of these aforementioned PTT agents. Medical University, #1 Panfu Road, Guangzhou 510180, China Superparamagnetic iron oxide nanoparticles (SPIONs), which possess unique Tel +86 20 8104 4902 properties including non-toxicity, magnetic properties, chemical stability, and Email [email protected] biocompatibility,9–11 have also been recently discovered to have photothermal effect and Li-Ming Zhang therefore could be used as an alternative PTT material.9,12–15 Chen et al14 reported that School of Materials Science and the highly crystallized iron oxide nanoparticles administered through the tail vein for Engineering, School of Chemistry, Sun Yat-sen University, #135 Xingang West PTT as effective and biodegradable mediators. These injected nanoparticles are likely to Road, Guangzhou 510275, China be applied for PTT as a result of the enhanced permeability and retention (EPR) effect Tel +86 20 8411 2454 Email [email protected] and is cleared from the body through urinary excretion without long-term toxicity. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2017:12 197–206 197 Dovepress © 2017 Yang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/IJN.S121249 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Yang et al Dovepress As already known, SPIONs were also used in clinical stud- CO2. Cells were both harvested by the use of trypsin and re- ies as an excellent T2-weighted magnetic resonance imaging suspended in a fresh complete medium before plating. (MRI) contrast agent. Hence, SPIONs basis theranostic agent, which could combine diagnostic MRI and NIR laser-induced Animals PTT therapeutic capabilities into one single entity, would be All animal experiments were performed under ethical approval a great value for optimal medical outcome.16–18 However, in from and in agreement with the guidelines of the Institutional many cases, nanoparticles are simultaneously transferred Animal Care and Use Ethics Committee of Sun Yat-sen to the reticuloendothelial system (RES) of liver and spleen, University and also in accordance with the policy of the National thus significantly reducing their treatment efficiency on Ministry of Health. Tumors were induced by subcutaneous injec- tumor cells. Therefore, there has been a growing demand tion of MDA-MB-231 cells (1×107 cells per mouse) into the right to develop various tumor-targeted SPIONs systems for the flank of BALB/c female nude mice (4–5 weeks, 18–20 g). PTT and MRI of tumor sites. A previous study19 developed a modified SPIONs Synthesis of HA-SPIONs naturally occurring glycosaminoglycan (hyaluronic acid, The hyaluronan-targeted SPIONs were synthesized 19 HA), with specific biological recognition to human cancers according to the method of Yang et al by the chemical con- overexpressing CD44 HA receptors, and this study set jugation of hyaluronan, as reported in a recent publication. out to evaluate the MRI ability and photothermal effect of HA-SPIONs which the previous study had developed for tar- Measurements of photothermal geted breast cancer to investigate its possibility to be used as a conversion effect theranostic agent. For this purpose, the photothermal conver- The dry powders obtained (10 mg) were placed on the sion effect was initially studied on dry HA-modified SPIONs parafilm (Polysciences Inc, Warrington, USA; thickness: powders. Then, HA-modified SPIONs were applied on CD44 0.127 mm). Lasers (Xi’an Minghui Optoelectronics Tech. HA receptors overexpressing MDA-MB-231 breast tumor Co., Ltd., Xi’an, China) with wavelength of 808 nm (power 2 cells to investigate the specific cellular uptake. Additionally, density, 1 W/cm ; spot area, 10×8 mm) were used to irradi- MRI and photothermal efficacy of HA-modified SPIONs ate the nanoparticle powders through the parafilm at room were investigated with in vitro and in vivo experiments. temperature. As a control, the parafilm was irradiated with the same lasers as described earlier. Experimental section To accurately monitor the temperature changes in samples Materials upon laser irradiation, the SPIONs and HA-SPIONs powders were dissolved in water (10 mL) in a round-bottomed flask to Ferric chloride hexahydrate (FeCl3⋅6H2O), ferrous get the nanoparticle aqueous suspension of different concen- sulfate (FeSO4), ammonium hydroxide, 1-ethyl-3-[3- (dimethylamino)-propyl]carbodiimide, N-hydroxysuccin- trations (10 mL, 100–500 μg/mL). The 808 nm lasers were imide, and fluorescein isothiocyanate were purchased from then used to irradiate the solutions for 1, 2, 3, 5, 7, or 9 min, Aladdin Reagent Database Inc. (Shanghai, China). HA respectively. As controls, the temperatures of distilled water (MW 320 kDa) was purchased from Freda Biochem Co., irradiated by the same laser as described earlier were mea- Ltd. (Shandong, China). 3-Aminopropyltrimethoxysilane sured. All the experiments were conducted in triplicate. (APTMS, pH 7.4) was purchased from Sigma-Aldrich (St Louis, MO). Fetal bovine serum (FBS, 10%), Dulbecco’s In vitro cellular uptake studies – Perl’s Modified Eagle’s Medium (DMEM) were purchased from Prussian blue staining 5 Hyclone/Thermo Scientific (Waltham, MA, USA). All other MDA-MB-231 cells at a density of 5×10 cells/well were seeded chemicals and reagents were of analytical grade. onto the 12-well plate and incubated for 24 h, and 1 mL of the DMEM containing SPIONs or HA-SPIONs (Fe concentration Cell culture was fixed to be 40μ g/mL) were added and incubated at 37°C for MDA-MB-231 cells, purchased from Land Biology Tech- 24 h. Then Perl’s Prussian blue staining assay was performed nology Company Co., Ltd. (Guangzhou, Guangdong, according to the method which was published earlier.19 The China), were cultured in DMEM supplemented with 10% wells without SPIONs or HA-SPIONs were used as control. of FBS, antibiotics (100 μg/mL streptomycin and 100 U/mL Perl’s Prussian