Accepted Manuscript Drug-loaded gold/iron/gold plasmonic nanoparticles for magnetic targeted chemophotothermal treatment of rheumatoid arthritis Hyung Joon Kim, Sun-Mi Lee, Kyu-Hyung Park, Chin Hee Mun, Yong-Beom Park, Kyung-Hwa Yoo PII:

S0142-9612(15)00468-8

DOI:

10.1016/j.biomaterials.2015.05.018

Reference:

JBMT 16855

To appear in:

Biomaterials

Received Date: 23 January 2015 Revised Date:

5 May 2015

Accepted Date: 14 May 2015

Please cite this article as: Kim HJ, Lee S-M, Park K-H, Mun CH, Park * Y-B, Yoo K-H, Drug-loaded gold/ iron/gold plasmonic nanoparticles for magnetic targeted chemo-photothermal treatment of rheumatoid arthritis, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Drug-loaded gold/iron/gold plasmonic nanoparticles

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for magnetic targeted chemo-photothermal treatment of rheumatoid arthritis

a

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Kyung-Hwa Yooa,c,*

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Hyung Joon Kima, Sun-Mi Leea, Kyu-Hyung Parkb, Chin Hee Munb, Yong-Beom Parkb,*, and

Nanomedical Graduate Program, Yonsei University, Seoul 120-749, Korea

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Division of Rheumatology, Department of Internal Medicine, Institute for Immunology and

Immunological Disease, Brain Korea 21 PLUS Project for Medical Science, Yonsei University,

Department of Physics, Yonsei University, Seoul 120-749, Korea

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Seoul 120-752, Korea

CORRESPONDING AUTHOR Tel.: +82-2-2123-3887

E-mail addresses: [email protected] (Y. B. Park), [email protected] (K. H. Yoo)

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ABSTRACT

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We have developed methotrexate (MTX)-loaded poly(lactic-co-glycolic acid, PLGA) gold (Au)/iron (Fe)/gold (Au) half-shell nanoparticles conjugated with arginine-glycine-aspartic acid (RGD), which can be applied for magnetic targeted chemo-photothermal treatment, and in vivo

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multimodal imaging of rheumatoid arthritis (RA). Upon near-infrared (NIR) irradiation, local heat is generated at the inflammation region due to the NIR resonance of Au half-shells and

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MTX release from PLGA nanoparticles is accelerated. The Fe half-shell layer embedded between the Au half-shell layers enables in vivo T2-magnetic resonance (MR) imaging in addition to NIR absorbance imaging. Furthermore, the delivery of the nanoparticles to the inflammation region in collagen-induced arthritic (CIA) mice, and their retention can be enhanced under external magnetic field. When combined with consecutive NIR irradiation and

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external magnetic field application, these nanoparticles provide enhanced therapeutic effects with an MTX dosages of only 0.05% dosage compared to free MTX therapy for the treatment of

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RA.

KEYWORDS: Au/Fe/Au plasmonic nanoparticles, magnetic targeting, dual in vivo imaging, chemo-photothermal treatment, rheumatoid arthritis

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1. Introduction

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Multifunctional nanoparticles (NPs) for multimodal imaging and therapy have been extensively studied for use in disease treatment [1]. Among them, near-infrared (NIR) resonant nanomaterials such as Au nanocages, Au nanoshells, Au nanorods, Au nanostars, carbon

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nanotubes, and Au nanoparticles coated with reduced graphene oxide strongly absorb light from the NIR spectrum and convert it into cytotoxic heat [2-7]. Thus, these materials have been

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widely studied for in vivo NIR absorbance imaging and photothermal therapy. Additionally, a combination of photothermal therapy, chemotherapy, and targeted delivery can have the synergistic effects for the treatment of cancer [8-13] and rheumatoid arthritis (RA) [14]. Here, we describe drug-loaded poly(lactic-co-glycolic acid, PLGA) gold (Au)/iron (Fe)/gold (Au) half-shell NPs conjugated with target moieties. NIR irradiation leads to local heat

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generation due to the NIR resonance of Au half-shells and accelerates MTX release from PLGA NPs. Furthermore, the Fe half-shell layer enables magnetic delivery of the NPs to the target

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region using a magnetic field, and their retention can be prolonged under an external magnetic field [15]. Thus, these NPs can be used for magnetic targeted chemo-photothermal treatments, as

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well as for dual imaging, such as magnetic resonance (MR) and NIR absorbance imaging. We have adopted these NPs to treat RA. RA is characterized by synovial inflammation of the joints [16], which are suitable for magnetic targeting and within the penetration depth of NIR. Disease-modifying anti-rheumatic drugs (DMARDs) are the main strategy for the treatment of RA, and methotrexate (MTX) is the most widely used DMARD [17]. However, long-term MTX administration may provoke adverse and dose-dependent effects, including stomatitis, alopecia, infection, liver toxicity, and bone marrow suppression [18]. Novel drug delivery systems aim to

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maintain a high MTX concentration in the joint spaces, while administering only small drug dosages and reducing side effects.

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For RA treatment, we loaded MTX inside PLGA NPs and conjugated arginine-glycineaspartic acid (RGD) peptide onto the Au surface to act as a targeting moiety for inflammation [19]. These NPs were injected intravenously into collagen-induced arthritic (CIA) mice, and

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were effectively delivered to the inflamed joints due to the RGD peptides as well as the leaky nature of the capillaries of RA [20]. Application of a magnetic field to the inflamed paw drew

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more NPs to the inflamed joints, where they accumulated for longer than 1 week, allowing two instances of exposure to NIR light, at 1 and 7 days after NP administration. Compared with conventional treatment, this magnetic targeted chemo-photothermal treatment provided superior

2.1. Materials

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2. Materials and Methods

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therapeutic efficacy with only 0.05% the dosage of MTX.

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Poly(lactic-co-glycolic acid) (PLGA; Mw = 20 k, 50:50) was purchased from Wako chemicals

(Japan).

Pluronic®

F-127,

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

hydrochloride (EDC), N-hydroxysuccinimide (NHS), and methotrexate were purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Thiol PEG Carboxylic acid (SH-PEGCOOH, Mw = 5 k) was purchased from Creative PEG-Works (USA). Deprotected cyclic RGDyK (cyclic Arg-Gly-Asp-D-Tyr-Lys, cyRGD) peptides were purchased from FutureChem

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Co. Ltd (Seoul, Korea), and Dulbecco’s phosphate buffered saline (PBS, pH 7.4) was purchased

2.2. Preparation of MTX-Au/Fe/Au plasmonic NPs

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from Hyclone (USA). All other chemicals, reagents, and solvents were analytical grade.

The fabrication process of MTX-Au/Fe/Au plasmonic NPs was shown in Fig. 1a. MTX-

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PLGA NPs were synthesized using the solvent evaporation method as previously reported [14]. Briefly, PLGA (200 mg) and MTX (6 mg) were dissolved in dichloroethane (20 ml), and this

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organic solution (oil phase) was added slowly drop-wise with distilled water (200 ml) containing Pluronic F-127 (200 mg) as a stabilizer under magnetic stirring. After mixing the oil and water phase, the mixture was emulsified by ultrasonication for 1 hr (400 W), followed by evaporation of organic solvent (dichloroethane) with stirring for 1 day. Then, the MTX-PLGA NPs were collected by centrifugation, and re-dispersed in 5 ml PBS by sonication. The electron-beam

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evaporator was used to deposit the films of 10-nm-thick Au/ 5-nm-thick Fe/ 10-nm-thick Au onto MTX-PLGA NPs monolayer, which was prepared by spin-coating aqueous suspension of

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MTX-PLGA NPs on a Si substrate. The Si substrate was immersed in SH-PEG-COOH (1 wt%) solution followed by sonication. After that, the COOH-terminated MTX-Au/Fe/Au plasmonic

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NPs were obtained by centrifugation. For targeted delivery to the inflammation region, the cyRGD peptides (3 mg) were conjugated on the outer Au half-shell surface of MTX-Au/Fe/Au plasmonic NPs with conventional EDC (4 mg) /NHS (4 mg) coupling reaction in 9 ml of PBS at 4 °C for 1 day. After the reaction, MTX-Au/Fe/Au plasmonic NPs were collected by centrifugation, and were re-dispersed in 1.5 ml of PBS. These cyRGD peptides conjugated on MTX-Au/Fe/Au plasmonic NPs were analyzed using a 1H-NMR spectrometer (Varian Gemini-300, DMSO-d6) with characteristic δ values of 1.5 (d, -

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CH3 of PLGA), 3.6 (s, -CH2- of PEG), 5.2 (m,

CH of PLGA), 8.1 - 8.6 (-NH(NH2)=NH of

cyRGD). In the FT-IR spectrum (Bruker Vertex 70), there were characteristic peaks at 1,700 cm(C=O stretch), 3,480 cm-1 (-N-H stretch), and 1,650 cm-1 (-N-H bend). The cyRGD peptides

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contents in the MTX-Au/Fe/Au plasmonic NPs were quantified by high pressure liquid

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chromatography (HPLC) using a C18 column (water Noca-Pak C18, 3.9 × 300 mm, 4 µm).

2.3. In vitro drug release

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The MTX-Au/Fe/Au plasmonic NPs (10 mg) were loaded into a dialysis tube (1 kDa cut-off membrane, Tube-O-DIALYZERTM, G-Biosciences, USA) followed by immersion in 10 ml PBS with mild constant shaking. During the dialysis, the solution volume was maintained constantly by replacing with fresh PBS after each sampling. The release experiments were conducted with

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and without two exposures to NIR light, with a laser diode of wavelength λ = 808 nm and beam diameter d ≈ 3.5 cm (Unique mode 30 k/400/20 (808 ± 3) nm, Jenoptik Co., Germany). NIR light of 0.62 W/cm2 was irradiated for 10 min at the start of the experiment at 37 oC and the second

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NIR was irradiated at 7 days. The amount of released MTX from the MTX-Au/Fe/Au plasmonic NPs was measured by ultraviolet-visible/NIR spectrophotometer at 304 nm. The measurements

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were performed three times for each sample.

2.4. Induction and treatment of collagen induced arthritis All procedures involving animals were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use

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Committee of Yonsei University Health System, Seoul, Korea. Male DBA/1J mice (8 weeks old; Central Lab Animal, Inc., Seoul, Korea) were injected intradermally at the tail base with 200 µg

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bovine type II collagen (CII; Chondrex, Redmond, WA, USA) emulsified in Freund’s complete adjuvant (1:1, v/v; Chondrex) containing 200 µg Mycobacterium tuberculosis H37Ra (Chondrex). Two weeks later, the mice were given intradermal booster injections of 100 µg CII

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in incomplete Freund’s adjuvant (1:1, v/v; Chondrex) [21].

Mice were monitored twice weekly for signs of arthritis onset based on paw swelling and

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clinical arthritis scoring on a scale of 0 – 4. Each paw was graded by two independent observers and the grades were summed, yielding a maximum possible arthritis score of 16 for each animal. Upon arthritis development (arthritis score of 8 - 10), mice were randomly arranged to each treatment group (n = 3) at 3 weeks after the second CII booster injection. Saline (G1) or MTXAu/Fe/Au plasmonic NPs (from G3 to G7) were administered via intravenous tail injection. G4,

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G6, and G7 were also exposed to 1.3 W/cm2 NIR light for 10 min at 1 day after NP injection. G7 received a second NIR exposure 7 days after the first. MTX solution (G2) with 35 mg/kg was

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injected intraperitoneally twice weekly till sacrifice. An external magnetic field was applied to

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G5, G6 and G7. After that, the mice were observed twice weekly during 31 days.

2.5. In vivo NIR imaging

Au/Fe/Au plasmonic NPs (150 µl, without MTX, 1 mg/ml in PBS) were injected intravenously to the CIA mice. The CIA mice were anesthetized by intraperitoneal injection of a Zoletil50/Rompun mixture (v/v = 3), and anesthesia was sustained during the experiments. NIR absorbance images were obtained for 7 days using the eXplore Optix System (Advanced Research Technologies Inc., Montreal, Canada). The total NIR absorbance intensities at the

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region-of-interest (ROI) in the inflamed paws of CIA mice were calculated using the eXplore

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Optix System software.

2.6. In vivo MR imaging

T2-weighted MR images were collected using a 9.4-T 20-cm-bore MRI system (Bruker

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Biospin, Ettlingen, Germany) equipped with a rat brain surface coil (4 ch). Animal imaging was performed under inhalational anesthesia with isofluorane (3% induction and 1% maintenance

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with 100% O2 at a 1,000 ml/min constant flow rate). T2-weighted MR images were acquired at 1 day and 7 days after intravenous injection of Au/Fe/Au plasmonic NPs (150 µl, without MTX, 1 mg/ml in PBS). T2 map and T2 color map images were processed with the Image Sequence

2.7. Histology study

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Analysis Tool of Bruker Paravision 5.1 (Bruker Biospin, Ettlingen, Germany).

The mice were sacrificed 32 days after each treatment and the joints were removed from the

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mice. The joints were fixed in buffered formalin-saline (10%) at 4 oC for 7 days and decalcified in formic acid (5%) in PBS for 3 weeks. The decalcified joints were embedded in paraffin blocks

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and paraffin sections were sliced (4-µm-thick). The joint tissues sections were stained with hematoxylin and eosin (H&E) and scored for changes in cell infiltration, bone erosion, and synovial proliferation, all on a scale of 0 - 4 [22]. Each score was assessed by two independent observers and the average grades were calculated. For histological examinations, the major organs from different groups of mice (G1 or G7) were harvested, and stained with H&E as described above.

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2.8. Biodistribution and clearance For quantitative analysis of biodistribution, the MTX-Au/Fe/Au plasmonic NPs (150 µl, 1

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mg/ml in PBS) were intravenously injected into CIA mice (n = 3), and the liver, spleen, and kidney were isolated from the mice at 1, 7, and 28 days post-injection. The tissues were dissolved in an aqua regia during overnight at 80 - 90 °C and re-heating at 120 – 130 °C was

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conducted for 12 h. The amount of ionized Au was analyzed by ICP-MS (PerkinElmer Nexion

2.9. Statistical analysis

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300S).

All experimental results were exhibited as the mean ± standard deviation and the Student’s t test was performed for the statistical analysis. Statistical significance was considered a value for

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3. Results and Discussion

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p < 0.05 (SAS 9.2.4, SAS Institute Inc.).

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3.1. Characterization of MTX-Au/Fe/Au Plasmonic NPs Fig. 1a shows the procedure for fabricating the RGD-conjugated MTX-loaded PLGA Au/Fe/Au half-shell NPs (MTX-Au/Fe/Au plasmonic NPs) used in our experiments. The size and zeta-potential of prepared nanoparticles dispersed in an aqueous medium were measured at 25 °C by using dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments Ltd.) as summarized in Table S1. Using dynamic light scattering, these NPs were determined to have an average diameter of ~135 nm and the estimated MTX loading content was about 2 wt%. The

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outer Au surface of the MTX-Au/Fe/Au plasmonic NPs was found to contain 19 µg immobilized RGD peptides/mg of NPs. The elemental mapping of an individual MTX-Au/Fe/Au plasmonic

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NP using TEM was shown is Fig. 1b. To test the stability of our NPs in in vitro condition, we measured the stability of NPs in 10% serum at 37 °C by measuring the absorbance of NPs solution at 800 nm as a function of time. The absorbance was nearly unchanged during 24 h in

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10% serum at 37 °C and did not exhibit any aggregation, indicating that the NPs were stable in in vitro condition (Fig. S1).

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Fig. 2a shows the ultraviolet-visible/NIR absorption spectra obtained from NPs with Au or Fe half-shell layers of different thicknesses. MTX-Au/Fe/Au plasmonic NPs with 10-nm Au/ 5nm Fe/ 10-nm Au half-shell thickness exhibited the most pronounced absorption peak at ~800 nm. NPs lacking the inner or outer Au layer showed absorption peaks at a wavelength longer

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than the biological optical window of 664 - 934 nm [23]. Thus, MTX-Au/Fe/Au plasmonic NPs were prepared with layers of 10-nm Au/5-nm Fe/10-nm Au. Finally, the cyRGD peptide targeting moiety was conjugated to SH-PEG-COOH modified on the outer Au surface, which

inflammation [19].

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binds αvβ3 integrins expressed on angiogenic vascular endothelial cells at regions of

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When a 0.5 mg/ml solution of MTX-Au/Fe/Au plasmonic NPs was irradiated by NIR light (λ = 808 nm, 0.62 W/cm2), the temperature increased to 55 °C within 10 min (Fig. 2b and Fig. S2), which is comparable to the reported results for Au nanorods [24] or nanoshells [25]. These results indicated that these NPs can be used for photothermal treatment and in vivo NIR absorbance imaging. The magnetic properties of MTX-Au/Fe/Au plasmonic NPs were also characterized by magnetization-magnetic field curve measurement. No hysteresis was observed, indicating that these NPs are superparamagnetic (Fig. 2c). To determine whether the NPs could

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be used as contrast for MR imaging, the spin-spin relaxation time (T2) and T2-weighted spin-echo MR images were measured at 1.5 T for NP solutions of various concentrations (Fig. 2d).

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Increasing NP concentrations were associated with increasing 1/T2, and these values were slightly higher than the 1/T2 values of commercially available contrast agent, Resovist, composed of Fe3O4 NPs [26]. These results indicated that MTX-Au/Fe/Au plasmonic NPs can be used as

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MRI contrast agents as well as NIR absorbance imaging agents.

Increasing temperature leads to more rapid degradation of PLGA-based NPs, and thus NIR

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irradiation increased the drug release rate from MTX-Au/Fe/Au plasmonic NPs. Fig. 3 shows the drug release profiles from MTX-Au/Fe/Au plasmonic NPs with two exposures to NIR light. The first 10-min NIR irradiation caused a 24-h burst release of MTX, followed by a slow release. The second NIR irradiation at 7 days induced another burst release of MTX. These results

NIR light.

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demonstrated the ability to control the MTX release from MTX-Au/Fe/Au plasmonic NPs using

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3.2. In vivo targeting efficacy

To evaluate the in vivo targeting efficacy, we generated collagen-induced arthritic (CIA)

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mice. These mice were injected intravenously with 150 µl of Au/Fe/Au plasmonic NPs (1 mg/ml stock solution, without MTX), and then placed in a mouse cage with or without the Nd-Fe-B magnet of 2.3 T (Fig. S3). The injected Au/Fe/Au plasmonic NPs were monitored by measuring time-lapse in vivo NIR images using an eXplore Optix System (Fig. 4a). Mice in the cage without the magnet (control group) showed increased absorbance intensity in the inflamed paws after NP injection, which slowly decreased over time, indicating NP delivery to and accumulation in the inflamed paws. In contrast, mice in the cage with the magnet exhibited

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continuously increasing absorbance intensity for up to 7 days, probably due to magnetic targeting and enhanced retention of the NPs (Fig. 4b). To quantitatively compare NP magnetic targeting

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and retention with and without magnetic field application, an inductively coupled plasma mass spectrometer (ICP-MS) was used to measure the Au accumulated in the inflamed joints (Fig. 4b). At 7 days after NP injection, the Au mass in the inflamed joints was ~ 0.934 µg (1.7% of the

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injected NPs) for mice in the cage without the magnet, and ~ 1.351 µg (2.4% of the injected NPs) for mice in the cage with the magnet. These findings support that magnetic field application

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enhanced the magnetic targeting and retention of Au/Fe/Au plasmonic NPs.

We also analyzed MR images of the CIA mice treated with Au/Fe/Au plasmonic NPs and cage with or without the magnet (Fig. S3). Since 1/T2 increased with increasing Au/Fe/Au plasmonic NPs concentration (Fig. 2d), lower T2 values represented more NPs. In the control

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group, similar amounts of NPs were accumulated in the inflamed paws at 1 and 7 days after NP injection. In contrast, the mice caged with the magnet showed larger NP amounts accumulated in the inflamed paw, and the NP amount increased over time (Figs. 5a and 5b), as seen in the in

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vivo NIR absorbance images. These results confirmed that external magnetic fields resulted in

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greater Au/Fe/Au plasmonic NP delivery to the inflamed paws and enhanced NP retention.

3.3. In vivo therapeutic effects of magnetic-targeted chemo-photothermal treatment in CIA mice To investigate the therapeutic effects of MTX-Au/Fe/Au plasmonic NPs, we conducted comparative efficacy studies (Fig. 6a and Fig. S4). The CIA mice were divided into seven groups (n = 3 in each group) and treated as summarized in Table 1. When arthritis was fully developed (group mean clinical index of 8 - 10), the mice were injected with saline (G1), MTX solution (G2), or MTX-Au/Fe/Au plasmonic NPs (G3-G7). G2 received twice weekly

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intraperitoneal injections of 35 mg/kg MTX solution till sacrifice. G3 through G7 received a single intravenous tail vein injection of NP solution containing 0.15 mg/kg of MTX (150 µl, 1

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mg/ml in PBS). G5, G6, and G7 were caged with the Nd-Fe-B magnet of 2.3 T (Fig. S3), while G3 and G4 were caged without the magnet. G4 and G6 were irradiated with NIR light (1.3 W/cm2) for 10 min at 1 day after injection. G7 was exposed to NIR light (1.3 W/cm2) for 10 min

temperature of the inflamed paw to ~ 45 °C (Fig. S5).

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on two occasions: at 1 and 7 days after injection (Fig. 3). NIR irradiation increased the

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Compared to saline-treated mice (G1), all other groups showed decreased clinical indices with some variations observed depending on the day. Photothermally treated mice (G4 and G6) generally exhibited lower clinical indices than mice treated with NPs and unexposed to NIR light (G3 and G5), regardless of magnetic field application. This implied that the MTX burst release upon NIR irradiation contributed to decreasing the clinical index more effectively compared to

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the slow MTX release without NIR irradiation. We also noted that the photothermally treated mice (G4 and G6; 0.15 mg/kg MTX), showed therapeutic efficacy comparable to the MTX

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treated mice (G2; 35 mg/kg × 9 times = 315 mg/kg), with only 0.05% of the MTX dosage used in G2. Interestingly, a second exposure to NIR light at the 7th day in the presence of external

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magnetic field (G7) achieved higher therapeutic effects. Thus, we may conclude that magnetic targeted chemo-photothermal treatment provides higher therapeutic efficacy compared with conventional or chemo-photothermal treatment. To confirm the therapeutic effects of the MTX-Au/Fe/Au plasmonic NPs combined with NIR irradiation and external magnetic field application, we performed histological examinations of joints at 32 days after intravenous injection (Fig. 6b). G1 showed inflammatory cell infiltration, bone erosion, and synovial proliferation, each of which was significantly decreased

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in G6 and G7. The results in G6 and G7 were comparable or superior to in the MTX treatment

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group (G2) (Fig. 6c).

3.4. In vivo toxicity of MTX-Au/Fe/Au plasmonic NPs

Histological examinations of major organs (liver, spleen, kidney, lung, and heart) were also

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performed at 32 days post-injection to investigate the influence of MTX-Au/Fe/Au plasmonic NPs on major organs (Fig. 7). Compared to the negative control, G7 showed no apparent tissue

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damage, implying that the NPs accumulated in major organs did not induce in vivo toxicity. We also performed hematological analyses, including white blood count (WBC), platelets, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine in blood (Table S2). Compared to the negative control, G3, G5, and G6 showed no

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significant toxicity supporting that the NPs caused no toxicity.

3.5. Biodistribution and clearance of MTX-Au/Fe/Au plasmonic NPs

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To investigate whether the accumulated MTX-Au/Fe/Au plasmonic NPs were cleared from the body, we used ICP-MS to measure Au in organs of mice caged with or without the magnet at

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1, 7, and 28 days after intravenous injection (Fig. 8). After 28 days, most MTX-Au/Fe/Au plasmonic NPs were cleared from the body regardless of external magnetic field application.

4. Conclusions In summary, the MTX-Au/Fe/Au plasmonic NPs were successfully fabricated and shown to be applicable for multimodal imaging and magnetic targeted chemo-photothermal treatment.

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Following intravenous injection of these NPs into CIA mice, in vivo NIR absorbance and MR images revealed NP accumulation in the inflamed paws, which was enhanced under the external

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magnetic field. NIR irradiation increased the temperature of the exposed area and accelerated the MTX release rate from the NPs, allowing the chemo-photothermal treatment. Furthermore, combination with consecutive NIR irradiation and external magnetic field application resulted in

ACKNOWLEDGMENT

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smaller MTX dosage (0.05%) in the injected NPs.

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higher therapeutic efficacy compared to conventional treatment, despite the use of a much

This research was financially supported by a grant from the Korean Health Technology R&D Project, Ministry of Health &Welfare (Grant No. A110905) and Ministery of Science, KT

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and Future Planning through the National Research Foundation (NRF) of Korea (Nos. 2011-

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0017486, 2012R1A4A1029061, and 2013R1A1A3A04009309).

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FIGURE CAPTIONS

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Fig. 1. (a) Schematic depiction of the process of fabricating MTX-Au/Fe/Au plasmonic NPs. (b) Elemental mapping of an individual MTX-Au/Fe/Au plasmonic NP using TEM.

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Fig. 2. (a) Visible/NIR absorption spectra of NPs with Fe and Au layers of different thicknesses. (b) Temperature versus time for the MTX-Au/Fe/Au plasmonic NPs and PBS with NIR light

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irradiation for 10 min. (c) Magnetization (M–H) curves measured for MTX-Au/Fe/Au plasmonic NPs. When the external magnetic field applied, the MTX-Au/Fe/Au plasmonic NPs moved toward magnet within 15 min (inset). (d) Magnetic resonance data of 1/T2 versus relative NP

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concentration. The inset shows T2-weighted images for different NP concentrations.

Fig. 3. Profiles of MTX release from MTX-Au/Fe/Au plasmonic NPs with and without NIR irradiation of 0.62 W/cm2 for 10 min at initial time and the second NIR was irradiated at 7 days.

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The temperature increased up to 45 °C after NIR irradiation. Data represent mean values for n =

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3, and the error bars represent standard deviation of the means.

Fig. 4. (a) Time-lapse in vivo NIR absorbance images of inflamed paws of CIA mice injected intravenously with Au/Fe/Au plasmonic NPs (150 µl, 1 mg/ml in PBS) and caged with (+) or without (−) a magnet. (b) Number of pixels in which the absorbance intensity was above 4 × 103 arbitrary unit as a function of time for Au/Fe/Au plasmonic NP-treated mice caged with (+) or without (−) a magnet (left axis). The right axis shows the amount of Au accumulated in the

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inflamed joints extracted from the Au/Fe/Au plasmonic NP-treated mice at 7 days after intravenous injection with (+) or without (−) a magnet. The Au mass was measured using ICP-

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MS. Error bars represent standard deviation (n = 3, *p < 0.05).

Fig. 5. (a) Time-lapse in vivo T2-weighted MR images of inflamed paws of CIA mice injected

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intravenously with Au/Fe/Au plasmonic NPs (150 µl, 1 mg/ml in PBS) with (+) or without (−) a magnet. (b) T2-weighted MR signal as a function of time for Au/Fe/Au plasmonic NPs-treated

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mice with (+) or without (−) a magnet.

Fig. 6. (a) Clinical index of the paws of CIA mice after treatment with saline (G1), MTX solution (35 mg/kg × 9 times; G2), or MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX; G3), as well as after treatment with MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX) followed

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by NIR irradiation (1.3 W/cm2, 10 min; G4), followed by caging with a magnet (2.3 T, 31 days; G5), followed by NIR irradiation and a the magnet (1.3 W/cm2, 10 min and 2.3 T, 31 days; G6),

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or followed by two exposure to NIR irradiation and a magnet (1.3 W/cm2 × 2 times, 10 min × 2 times and 2.3 T, 31 days; G7). Error bars represent the standard deviation (n = 3). *p < 0.005

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compared to the saline treated mice (G1). (b) Histology of joint tissues extracted from a normal mouse (NC) and from CIA mice 32 at days after each treatment. H&E-stained representative joint sections from the experiment are shown (synovial inflammation, original magnification, 100×). (c) Semiquantitative analysis of histopathological evaluation (cell infiltration, bone erosion, and synovial proliferation) of CIA mice. Error bars represent the standard deviation (n = 3). *p < 0.05 compared to the saline treated mice (G1).

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Fig. 7. Histological sections of major organs extracted 32 days after intravenous injection of saline (top) or MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX) with repeated NIR

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irradiation and under the magnet (1.3 W/cm2 × 2 times, 10 min × 2 times and 2.3 T, 31 days, G7) (bottom). H&E-stained images were acquired at 400× magnification.

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Fig. 8. Tissue distribution of NPs in major organs (liver, spleen, and kidney) at different times. The amount of Au was measured by ICP-MS. Error bars represent standard deviation (*p < 0.05,

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n = 3).

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[23] Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19:316-7. [24] Hong YC, Lee EG, Choi JH, Oh SJ, Haam SJ, Huh YM, et al. Gold nanorod-mediated

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SYNOPSIS TOC

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Table 1. Summary of the treatments applied to CIA mice to compare therapeutic efficacy. Injected contenta

Dosage of MTX (mg/kg)

NIR light (W/cm2)b

Magnetic field (T)c

1

Saline

-

-

-

2

MTX solution

35 × 9 times

3

MTX-Au/Fe/Au plasmonic NPs

0.15

4

MTX-Au/Fe/Au plasmonic NPs

0.15

5

MTX-Au/Fe/Au plasmonic NPs

0.15

6

MTX-Au/Fe/Au plasmonic NPs

0.15

7

MTX-Au/Fe/Au plasmonic NPs

0.15

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Group (n = 3)

-

-

1.3

-

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-

-

2.3

1.3

2.3

1.3 × 2 times

2.3

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a

-

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Administration was performed by intravenous tail vein injection of 150 µl of 1 mg/ml nanoparticle concentration. b The arthritis joint was exposed to NIR light with a laser diode (λ = 808 nm) for 10 min at 24 h post-injection. In group 7, this was repeated 7 days later. c The external magnetic field was applied for up to 31 days starting immediately after injection.

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Fig. 1. (a) Schematic depiction of the process of fabricating MTX-Au/Fe/Au plasmonic NPs. (b)

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Elemental mapping of an individual MTX-Au/Fe/Au plasmonic NP using TEM.

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Fig. 2. (a) Visible/NIR absorption spectra of NPs with Fe and Au layers of different thicknesses. (b) Temperature versus time for the MTX-Au/Fe/Au plasmonic NPs and PBS with NIR light

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irradiation for 10 min. (c) Magnetization (M–H) curves measured for MTX-Au/Fe/Au plasmonic

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NPs. When the external magnetic field applied, the MTX-Au/Fe/Au plasmonic NPs moved toward magnet within 15 min (inset). (d) Magnetic resonance data of 1/T2 versus relative NP concentration. The inset shows T2-weighted images for different NP concentrations.

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Fig. 3. Profiles of MTX release from MTX-Au/Fe/Au plasmonic NPs with and without NIR irradiation of 0.62 W/cm2 for 10 min at initial time and the second NIR was irradiated at 7 days.

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The temperature increased up to 45 °C after NIR irradiation. Data represent mean values for n =

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3, and the error bars represent standard deviation of the means.

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Fig. 4. (a) Time-lapse in vivo NIR absorbance images of inflamed paws of CIA mice injected intravenously with Au/Fe/Au plasmonic NPs (150 µl, 1 mg/ml in PBS) and caged with (+) or without (−) a magnet. (b) Number of pixels in which the absorbance intensity was above 4 × 103

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arbitrary unit as a function of time for Au/Fe/Au plasmonic NP-treated mice caged with (+) or without (−) a magnet (left axis). The right axis shows the amount of Au accumulated in the

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inflamed joints extracted from the Au/Fe/Au plasmonic NP-treated mice at 7 days after intravenous injection with (+) or without (−) a magnet. The Au mass was measured using ICP-

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MS. Error bars represent standard deviation (n = 3, *p < 0.05).

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Fig. 5. (a) Time-lapse in vivo T2-weighted MR images of inflamed paws of CIA mice injected intravenously with Au/Fe/Au plasmonic NPs (150 µl, 1 mg/ml in PBS) with (+) or without (−) a

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magnet. (b) T2-weighted MR signal as a function of time for Au/Fe/Au plasmonic NPs-treated

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mice with (+) or without (−) a magnet.

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Fig. 6. (a) Clinical index of the paws of CIA mice after treatment with saline (G1), MTX solution (35 mg/kg × 9 times; G2), or MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX; G3),

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as well as after treatment with MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX) followed by NIR irradiation (1.3 W/cm2, 10 min; G4), followed by caging with a magnet (2.3 T, 31 days; G5), followed by NIR irradiation and a the magnet (1.3 W/cm2, 10 min and 2.3 T, 31 days; G6),

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or followed by two exposure to NIR irradiation and a magnet (1.3 W/cm2 × 2 times, 10 min × 2 times and 2.3 T, 31 days; G7). Error bars represent the standard deviation (n = 3). *p < 0.005

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compared to the saline treated mice (G1). (b) Histology of joint tissues extracted from a normal mouse (NC) and from CIA mice 32 at days after each treatment. H&E-stained representative joint sections from the experiment are shown (synovial inflammation, original magnification, 100×). (c) Semiquantitative analysis of histopathological evaluation (cell infiltration, bone erosion, and synovial proliferation) of CIA mice. Error bars represent the standard deviation (n =

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3). *p < 0.05 compared to the saline treated mice (G1).

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Fig. 7. Histological sections of major organs extracted 32 days after intravenous injection of saline (top) or MTX-Au/Fe/Au plasmonic NPs (0.15 mg/kg of MTX) with repeated NIR irradiation and under the magnet (1.3 W/cm2 × 2 times, 10 min × 2 times and 2.3 T, 31 days, G7)

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(bottom). H&E-stained images were acquired at 400× magnification.

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Fig. 8. Tissue distribution of NPs in major organs (liver, spleen, and kidney) at different times. The amount of Au was measured by ICP-MS. Error bars represent standard deviation

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(*p < 0.05, n = 3).

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ACCEPTED MANUSCRIPT Supplementary data

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Drug-loaded gold/iron/gold plasmonic nanoparticles for magnetic targeted chemo-

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photothermal treatment of rheumatoid arthritis

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Hyung Joon Kima, Sun-Mi Leea, Kyu-Hyung Parkb, Chin Hee Munb, Yong-Beom Parkb,*, and Kyung-Hwa Yooa,c,*

a

Nanomedical Graduate Program, Yonsei University, Seoul 120-749, Korea Division of Rheumatology, Department of Internal Medicine, Institute for Immunology and

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b

Immunological Disease, Brain Korea 21 PLUS Project for Medical Science, Yonsei

Department of Physics, Yonsei University, Seoul 120-749, Korea

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University, Seoul 120-752, Korea

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ACCEPTED MANUSCRIPT Table S1. Characterization of Prepared Nanoparticles. Au/Fe/Au Thickness (nm)

RGD content (µg/mg of Nanoparticles)e

-

-

10/5/10

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Nanoparticles

Size (nm) a

PDIb

MTX-PLGA Nanoparticles

105

0.102

-40.92 ± 2.07

3.49 ± 0.05

135

0.197

-38.50 ± 1.52

2.05 ± 0.13

MTXAu/Fe/Au

Plasmonic

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Nanoparticles

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MTX loading content (wt%)d

Zetapotential (mV)c

a,b,c

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The size, polydispersiy index (PDI) and zeta-potential of the nanoparticles were measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments Ltd) at a concentration of approximately 1 mg of nanoparticles/1 ml of distilled water.

d

To determine the amount of encapsulated MTX, the dried nanoparticles were redissolved in DMSO to extract MTX form the nanoparticles. MTX in the supernatant was quantified by a UV-VIS-NIR spectrometer (Cary 5000, VARIAN).

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The amount of grafted RGD in the nanoparticles was quantified by a HPLC using a C18 column (water Noca-Pak C18, 3.9 × 300 mm, 4 µm).

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Negative control

WBC (K/ul)

4,000 ~ 15,000

4,240

4,650

Platelets 3 (10 K/ul)

600 ~ 1,000

606

812

AST (U/l)

54 ~ 298

81

86

ALT (U/l)

17 ~ 77

BUN (mg/dl)

8 ~ 33

Creatinine (mg/dl)

0.2 ~ 0.9

MTXAu/Fe/Au Plasmonic NPs + Magnet + NIR (G6)

4,055

4,013

904

792

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Reference range

MTXAu/Fe/Au Plasmonic NPs + Magnet (G5)

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Parameters

MTXAu/Fe/Au Plasmonic NPs (G3)

121

123

31

50

29

25.3

18.3

16.0

19.1

0.46

0.20

0.20

0.20

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WBC : white blood count, AST : aspartate aminotransferase, ALT : alanine aminotransferase, BUN : blood urea nitrogen.

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Fig. S1. The stability of MTX-Au/Fe/Au plasmonic NPs in 10% serum at 37 °C.

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Fig. S2. The change of temperature increase of MTX-Au/Fe/Au plasmonic NPs for five

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NIR laser diode on/off cycles (λ = 808 nm, 0.62 W/cm2, beam diameter d ≈ 3.5 cm).

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Fig. S3. Photo of magnet cart of CIA mice for the application of magnetic field during

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experiments. The magnet was placed under CIA mice cage.

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Fig. S4. Photo of paws from the CIA mice before (0 d) and after treatment (31 d) with

saline (G1), MTX solution (35 mg/kg × 9 times, G2), and MTX-Au/Fe/Au plasmonic NPs

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(0.15 mg/kg of MTX) with repeated NIR irradiation and under the magnet (1.3 W/cm2 × 2 times, 10 min × 2 times and 2.3 T, 31 days, G7). The same mouse of each group (G1, G2, and

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G7) was observed during 31 days.

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Fig. S5. Thermal images of CIA mice treated with saline, MTX-Au/Fe/Au plasmonic N

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Ps (150 µl, 1 mg/ml in PBS) before and after NIR irradiation (1.3 W/cm2, 10 min) of the inflamed paws. The thermal images of MTX-Au/Fe/Au plasmonic NPs injected CIA m ouse for NIR irradiation were acquired using thermal imaging camera (FLIR-T250, FL

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IR, Sweden).

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gold plasmonic nanoparticles for magnetic targeted chemo-photothermal treatment of rheumatoid arthritis.

We have developed methotrexate (MTX)-loaded poly(lactic-co-glycolic acid, PLGA) gold (Au)/iron (Fe)/gold (Au) half-shell nanoparticles conjugated with...
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