Magnetic Targeting Enhanced Theranostic Strategy Based

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agnetic Targeting Enhanced Theranostic Strategy Based Mon Multimodal Imaging for Selective Ablation of Cancer Zhiwei Li ,Shengnan Yin ,Liang Cheng ,Kai Yang ,Yonggang Li ,and Zhuang Liu *of the theranostic agent to targeted lesions such as tumors, multimodal imaging contrasts to offer detailed tumor charac-teristics and enable post-therapeutic reex-aminations under imaging techniques, highly ef? cient and controllable tumor therapy, as well as the satisfactory bio-safety of the nano-agent. The ef? cient delivery and speci? c tar-geting of theranostic agents to the tumor region are utmost important in cancer [2treatment. ] A great deal of efforts has been paid to exploit various strategies to deliver theranostic agents to tumors with high ef? ciency and speci? city. For example, the enhanced permeability and retention effect (EPR) of cancerous tumors could allow passive tumor accumulation of nanoparticles with appropriate sizes [3and surface coatings. ] However, such a passive tumor targeting strategy suffers from the pathophysiological heteroge-[4neity of tumors. ] Molecular targeting is another well-established method relying the speci? c binding between targeting ligands conjugated on the nanoparticle 1.Introduction surface and receptors over-expressed on the membrane of tumor cells or tumor vasculature cells. Unfortunately, despite As the ? eld of nanomedicine has emerged from the blending the tremendous successes of molecular tumor targeting in of nanoscience and biomedicine, theranostics with concurrent many pre-clinical tumor models, its ef? ciency in real clinical and complementary diagnostic and therapeutic capabilities cases has been largely limited by the signi? cant inter-patient based on nanotechnology offers great opportunities in the ? ght [5]variations of receptor expressions. In recent years, a number [1against cancer. ] In an effort to realize the theranostic concept, of other tumor targeting approaches utilizing physical forces or the nano-platform should be deliberately engineered to simulta-stimulus, such as magnetic ? eld (MF), light, and ultra-sound, neously ful? ll a number of features, including speci? c delivery to enable tumor speci? c delivery of therapeutic agents have [6,7]received signi? cant interests, as physical interactions are Z. W. Li, Dr. L. Cheng, K. Yang, Prof. Z. Liu independent of the complicated cancer molecular biology path-Institute of Functional Nano & ways and their effects are more controllable and predictable. Soft Materials (FUNSOM) Among them, magnetic tumor targeting which uses magnetic Soochow University Suzhou, 215123, China nanoparticles carrying therapeutic functions and an external -mail: zliu@http://wendang.chazidian.com Emagnetic ? eld focused on the targeted tumor region, has Z. W. Li, Dr. L. Cheng, K. Yang, Prof. Z. Liu emerged as a promising approach that greatly overcomes limi-Collaborative Innovation Center of tations of molecular tumor targeting without being constrained Suzhou Nano Science and Technology [6,8]by the speci? c receptor expression. Soochow University Suzhou ,215123 ,China In this work, we design a magnetic targeting enhanced thera- S. N. Yin, Dr. Y. G. Li nostic strategy based on MR/PA multimodal imaging for selec-Department of Radiology tive photothermal ablation of tumors using gold shelled iron The First Af? liated Hospital of Soochow University oxide nanoclusters with polyethylene glycol coating (IONC@Suzhou ,Jiangsu ,215006 ,China Au-PEG). Such composite nanoparticles exhibit strong mag-netic property and high near-infrared (NIR) optical absorbance, DOI: 10.1002/adfm.201303345 The booming development of nanomedicine offers great opportunities for cancer diagnostics and therapeutics. Herein, a magnetic targeting-enhanced cancer theranostic strategy using a multifunctional magnetic-plasmonic nano-agent is developed, and a highly effective in vivo tumor photothermal therapy, which is carefully planed based on magnetic resonance (MR)/photoacoustic (PA) multimodal imaging, is realized. By applying an external magnetic ? eld (MF) focused on the targeted tumor, a magnetic targeting mediated enhanced permeability and retention (MT-EPR) effect is observed. While MR scanning provides tumor localization and reveals time-dependent tumor homing of nanoparticles for therapeutic planning, photoacoustic imaging with higher spatial resolution allows noninvasive ? ne tumor margin delineation and vivid visualization of three dimensional distributions of theranostic nanoparticles inside the tumor. Utilizing the near-infrared (NIR) plasmonic absorbance of those nanoparticles, selective photothermal tumor ablation, whose ef? cacy is predicted by real-time infrared thermal imaging intra-therapeutically, is carried out and then monitored by MR imaging for post-treatment prognosis. Overall, this study illustrates the concept of imaging-guided MF-targeted photo-thermal therapy based on a multifunctional nano-agent, aiming at optimizing therapeutic planning to achieve the most ef? cient cancer therapy. http://wendang.chazidian.com© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimAdv. Funct. Mater. 2014, 24, 2312–2321

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and thus offer great contrasts in both magnetic resonance (MR) imaging, a traditional imaging approach widely used in

[9the clinic for whole-body imaging, ] and photoacoustic (PA)

imaging, a recently emerged technique that allows higher-res-[10–12]olution imaging within a depth of a few cm. In our study,

both MR and PA imaging are carried out, not only to visualize the tumor, but also to determine the ef? ciency of magnetic tumor-targeting in a time-dependent manner for better thera-peutic planning. Owing to the strong magnetism of theranostic nanoparticles, remarkably improved tumor homing of those nanoparticles is observed under magnetic targeting, likely owing to the “magnetic targeting mediated EPR effect”. Photo-thermal treatment of cancer is then carefully planned and car-ried out. By ? nally tuning the laser power density and closely monitoring the tumor temperature, selective ablation of tumors under magnetic targeting is demonstrated. Moreover, at the post-treatment stage, imaging is further performed for tumor prognosis to evaluate the therapeutic outcome ( Figure 1). Our work deliberately demonstrates the unique advantages of multi-modal imaging guided therapeutic planning and post-treatment monitoring based on multifunctional theranostic nano-agents.

rather uniform sizes with an average diameter of ≈100 nm

(Figure 2 b,c and Supporting Information, Figure S2a). Dopa-[14mine (DA) was introduced to modify IONCs, ] making those nano-clusters water-soluble and positively charged with a zeta potential of +19 mV (Supporting Information, Figure S2b), which facilitated the adsorption of negative-charged gold seeds

[15through the electrostatic interaction. ] Afterwards, an in-suit

seed-mediated gold growth was carried out by reduction of

[16]HAuCl 4 to form a dense gold shell outside IONCs. Transmis-sion electron microscopy (TEM) images of nano-clusters before

and after gold growth witnessed the formation of a dense gold shell with seeds (small dark dots) embedded on top of IONCs. The core/shell multi-component structure of IONC@Au was further con? rmed by the high-angle annular dark ? eld scan-ning TEM (HAADF-STEM) images (Figure 2 d) and energy-dispersive X-ray spectroscopy (EDS) (Figure 2 e), both of which revealed the uniform coating of Au on IONCs. After the growth of gold shell, the obtained IONC@Au was then modi? ed by lipoic acid terminated PEG (LA-PEG, 5 kDa) through the gold-thiol bond. The successful PEGylation was evidenced by the infrared (IR) spectra as well as the remarkably enhanced physi-ological stability of IONC@Au-PEG (Supporting Information, Figure S3a,b).

By adjusting the added volume of gold growth solution, the

2.Results and discussion

gold shell thickness as well as the absorbance of IONC@Au-PEG composites could be controlled. UV/Vis/NIR spectra of IONC@The fabrication of IONC@Au and the subsequent function-

alization with polyethylene glycol (PEG) was illustrated in Au-PEG after adding different volumes of the growth solution Figure 2 a. IONCs were synthesized using a binary solvent showed the enhanced NIR absorbance of the sample as the thermal method according to the literature protocol with slight volume increase of HAuCl 4 growth solution from 5 mL to 50 mL

[13]2 f), consistent to the increased gold shell thickness as modi? cations. Being clusters of nanocrystals (Supporting (Figure

Information, Figure S1), the as-made IONCs showed revealed by TEM images (Supporting Information, Figure S4).

However, further increase of HAuCl 4volume from 50 mL to 100 mL resulted in obvious aggregation of composite nanoparticles (Sup-porting Information, Figure S4), and offered little additional enhancement of NIR absorb-ance. Consequently, we chose the sample pre-pared by adding 50 mL of growth solution (the optimized synthesis condition) for the pre-vious materials characterization (Figure 2 b-e, Supporting Information, Figures S2,S3) and the followed experiments. The precise molar ratio of Fe /Au in this nanocomposite was 3O4

measured to be 2.55:1 by inductively cou-pled plasma atomic emission spectroscopy (ICP-AES).

Next, the magnetic properties of IONCs

and IONC@Au-PEG were studied. The ? eld-dependent magnetization measure-ment suggested the superparamagnetic

Figure 1. The magnetic targeting enhanced theranostic strategy using IONC@Au-PEG nano-nature of IONCs before and after gold

coating (Figure 2g ). While being well dis-particles under guidance by multimodal imaging. In our experiment, IONC@Au-PEG is intra-venously injected into a mouse bearing two tumors, one of which is exposed to an external persed in water without any aggregation,

magnetic ? eld while the other is not. As the theranostic nanoparticles circulate in bloodstream, IONC@Au-PEG once exposed to an external they will be trapped into the magnetic ? eld created by the nearby magnet, resulting in enhanced magnetic ? eld could be rapidly attracted enrichment and prolonged retention in the targeted tumor. Dual modal MR and photoacoustic

by the magnet (Figure 2 g, inset). With a imaging is carried out to track and understand the tumor homing of our theranostic nanopar-ticles for therapeutic planning. IR thermal imaging is conducted during NIR laser irradiation concentration-dependent darkening effect

to real-time monitor the photothermal effect for better therapeutic control. MR imaging after in T2-weighted MR images, the T2 relax-photothermal therapy is ?

nally performed for post-treatment prognosis. ivity of IONC@Au-PEG was determined to

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Figure 2. Preparation and characterization of IONC@Au-PEG. a) A simpli? ed procedure for the fabrication of IONC@Au-PEG theranostic nanopar-ticles. b) SEM images of IONCs (left) and IONC@Au-PEG (right). c) TEM images of IONCs (left) and IONC@Au-PEG (right). d) STEM image and HAADF-STEM-EDS mapping images of IONC@Au-PEG. e) Cross-sectional compositional line pro? le in STEM pattern highlighted in (d). f) UV-Vis-NIR spectra of IONC@Au-PEG prepared by adding different volumes of gold growth solution. g) Magnetization loops of IONCs and IONC@Au-PEG. Inset: photos of IONC@Au-PEG aqueous solution without (left) and with (right) exposure to an external magnetic ? eld. h) The T2-weighted MR images of

IONC@Au-PEG under various Fe concentration and the relative relaxation rate R2.

be 88.01 m M?1 S ?1 (in H2 O at a ? eld strength of 3 T at room temperature) (Figure 2 h), suggesting the feasibility of using our composite nanoparticles as a contrast agent in MR imaging. The bio-compatibility and safety of nano-agents are one of

the main concerns in the area of nanomedicine. As ferrite nanoparticles could gradually decomposed into iron ions and several formulations of functionalized iron oxide nanoparticles have already been used for clinical MR diagnosis for many years, they have been proven to be biocompatible after admin-[17]istration. Besides, it has been evidenced that the chemical

inertness nature of gold nanomaterials renders them low tox-[18,19icity in biological systems. ] As for PEGylated IONC@Au

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core/shell nanocomposite developed in this work, their in vitro

cytotoxicity was ? rstly determined by the standard methyl thia-zolyl tetrazolium (MTT) cell viability assay ( Figure 3a), which indicated no obvious cytotoxicity with the nanoparticle concen-tration increased up to 0.4 mg/mL. Furthermore, lactate dehy-drogenase (LDH) leakage and intracellular reactive oxygen spe-cies (ROS) generation were examined to evaluate the possible cell membrane damage and oxidative stress of cells induced by IONC@Au-PEG, respectively. Our data revealed that those nanoparticles even at the highest tested concentration resulted in neither appreciable LDH leakage nor oxidative stress to cells (Figure 3b).

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Figure 3. In vitro experiments. a) Relative cell viability data determined by the MTT assay after culturing 4T1 cells with IONC@Au-PEG at various concentrations for 24 h. b) Data of LDH leakage and ROS generation assays of cells after incubation with IONC@Au-PEG under various concentra-tions for 24 h. Triton X-100 and H 2O2 were added as the positive controls for LDH and ROS assays, respectively. c) Photothermal heating curves of

–2 , 5 min). Inset: IR thermal imaging of water (left), IONCs (middle) and water, IONCs and IONC@Au-PEG under laser irradiation (808 nm, 1 W cm

IONC@Au-PEG (right) solutions before and after laser irradiation. d) The concentration-dependent in vitro photothermal therapy after laser irradiation

–2 (808 nm, 1 W cm , 5 min). The relative cell viabilities after various treatments were determined by the MTT assay. e) Confocal ? uorescence images of

–2Calcein AM and PI co-stained 4T1 cells after MF-targeted in vitro photothermal therapy irradiated by a laser (808 nm, 1 W cm , 5 min). Images were

taken at different locations in the culture dish: 1) far from the magnet, 2) in the middle, and 3) nearby the magnet.

ext, we tested the photothermal performance of IONC@N

Au-PEG nanoparticles under NIR laser exposure. Owing to the

high NIR absorbance, an aqueous solution of IONC@Au-PEG

–1 (1 mg mL ) could be rapidly heated up to ≈57 °C after being irra-

–2for diated by an 808-nm laser under a power density of 1 W cm

5 min (Figure 3 c). In contrast, IONCs without gold coating

showed much less effective photothermal heating because of

their low absorbance at 808 nm. The photothermal effect induced

tumor cell death was then demonstrated with 4T1 cells. As

expected, nanoparticle concentration-dependent photothermal destruction of cancer cells was observed, with the majority of cells –1 of IONC@Au-PEG and killed after incubation with 0.1 mg mL –2 for 5 min (Figure exposed to the 808-nm laser at 1 W cm 3 d). To demonstrate the in vitro magnetic ? eld (MF) controlled photo-thermal cancer ablation, 4T1 cells were incubated with IONC@Au-PEG in a cell culture dish, beneath which a magnet was placed. Owing to the localized accumulation of IONC@Au-PEG nanoparticles induced by the MF, only those cells nearby the magnet were effective ablated by the NIR laser (808 nm, –2 1 W cm , 5 min), leaving the rest cells untouched (Figure 3 e).

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Figure 4. In vivo MR/photoacoustic dual-modal imaging. a) T2-weighted in vivo MR images acquired at different time intervals post injection of IONC@Au-PEG. A much more remarkable time-dependent darkening effect in MF-targeted tumor (on the right) than that in the non-targeted (on the left) was observed. b) 2D and 3D photoacoustic imaging of tumors taken 24 hours after injection of IONC@Au-PEG. From left to right: PA imaging of control, non-targeted and MF-targeted tumor. c) T2 signal intensities and relative T2-signal change (?T2/T2%) of tumors at different time intervals post injection. d) Quanti? cation of MR and photoacoustic signals in tumors 24 hours post injection of IONC@Au-PEG. e) Study of long-term tumor retention of IONC@Au-PEG under magnetic targeting for 1 day (upper) or 7 days (lower) by MR imaging. White and red dashed circles indicate tumors in the absence and presence of magnetic targeting, respectively. f) The quantitative T2 signal changes at different time intervals corresponding to images in (e). P values were calculated by student’s t-test: *** P < 0.001, ** P < 0.01.

e then wondered whether the external magnetic ?W eld would guide the tumor speci? c homing of PEGylated IONC@Au nanoparticles. Balb/c mice each bearing two 4T1 tumors were intravenously (i.v.) injected with IONC@Au-PEG (200 μL,

–12 mg mL ) through the tail vein. To enable magnetic targeting,

a magnet was securely attached onto the right tumor of each mouse by a bandage all the time in our experiments except being shortly taken off during imaging. Those mice were then imaged by a 3 T MR scanner at various time intervals post injection (p.i.) ( Figure 4 a). Both MF-targeted and non-targeted tumors showed obvious time-dependent darkening effects in T2-weighted MR images post injection of IONC@Au-PEG, indicating the passive tumor accumulation of nanoparticles as a result of the tumor EPR effect. Notably, compared to the non-targeted tumors (white dashed circles and arrows), the MF-targeted tumors (red dashed circles & arrows) appeared to be much darker in T2-MR images, highlighting the remarkably enhanced tumor homing of our theranostic nanoparticles under magnetic targeting.

Photoacoustic imaging is able to detect ultrasound waves

generated from thermally induced expansion and vibration

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[12of light absorbing tissues under pulsed laser irradiation. ]

While MR imaging is capable of whole-body imaging without

[20any depth limit, ] photoacoustic imaging offers much higher

spatial resolution and could image tissues in a localized region

[11,21with a maximal depth of 5–7 cm. ] Thus, in our study, PA

imaging was carried out, in order to allow ? ne margin deline-ation of the tumor, visualize the tumor micro-structures (e.g., vasculature structures), and understand the intratumor dis-tribution of IONC@Au-PEG nanoparticles at a higher spatial resolution (Figure 4 b,c). While both 2D and 3D rendering of PA images revealed the major vasculatures in the tumor of a mouse prior to the injection of nanoparticles, strong PA signals which were mainly associated with the tumor vasculatures were observed in the MF-targeted tumor of this mouse 24 h after injection with IONC@Au-PEG. The non-targeted tumor, on the other hand, showed much weaker PA signals, consistent to the MR imaging data. Therefore, we could conclude that the external MF is able to attract IONC@Au-PEG magnetic nano-particles circulating in the blood vessels to the tumor region, where the vasculatures are disordered and damaged, leading

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