Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery.

Acta Biomaterialia xxx (2016) xxx–xxx

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Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery Dai Hai Nguyen a,1, Jung Seok Lee b, Jong Hoon Choi a, Kyung Min Park c, Yunki Lee a, Ki Dong Park a,⇑ a b c

Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong, Suwon 443-749, Republic of Korea Biomedical Engineering, Yale University, CT 06511, USA Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

a r t i c l e

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Article history: Received 9 November 2015 Received in revised form 11 February 2016 Accepted 12 February 2016 Available online xxxx Keywords: Self-assembled magnetic nanoclusters Superparamagnetic iron oxide Cyclodextrin Poly(ethylene glycol) Paclitaxel MRI Controlled drug delivery Theranostics

a b s t r a c t Nanoparticle-based imaging and therapy are of interest for theranostic nanomedicine. In particular, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) have attracted much attention in cancer imaging, diagnostics, and treatment because of their superior imagability and biocompatibility (approved by the Food and Drug Administration). Here, we developed SPIO nanoparticles (NPs) that self-assembled into magnetic nanoclusters (SAMNs) in aqueous environments as a theranostic nano-system. To generate multi-functional SPIO NPs, we covalently conjugated b-cyclodextrin (b-CD) to SPIO NPs using metaladhesive dopamine groups. Polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the b-CD cavity through high affinity complexation. The core-shell structure of the magnetic nanoclusters was elucidated based on the condensed SPIO core and a PEG shell using electron microscopy and the composition was analyzed by thermogravimetric analysis (TGA). Our results indicate that nanocluster size could be readily controlled by changing the SPIO/PEG ratio in the assemblies. Interestingly, we observed a significant enhancement in magnetic resonance contrast due to the large cluster size and dense iron oxide core. In addition, tethering a tumor-targeting peptide to the SAMNs enhanced their uptake into tumor cells. PTX was efficiently loaded into b-CDs and released in a controlled manner when exposed to competitive guest molecules. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy. Statement of Significance In this study, we developed multi-functional SPIO NPs that self-assembled into magnetic nanoclusters (SAMNs) in aqueous conditions as a theranostic nano-system. The beta-cyclodextrin (b-CD) was immobilized on the surfaces of SPIO NPs and RGD-conjugated polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the b-CD cavity through high affinity complexation. We found that nanocluster size could be readily controlled by varying the SPIO/PEG ratio in the assemblies, and also demonstrated significant improvement of the functional nanoparticles for theranostic systems; enhanced magnetic resonance, improved cellular uptake, and efficient PTX loading and sustained release at the desired time point. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy. Ó 2016 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

1. Introduction

⇑ Corresponding author at: Department of Molecular Science and Technology, Ajou University, Padal Hall #504, Suwon 443-749, Republic of Korea. E-mail address: [email protected] (K.D. Park). 1 Current address: Institute of Applied Materials Science, Vietnam Academy Science and Technology, 1 Mac Dinh Chi, Ho Chi Minh City, Viet Nam.

Over the past decades, advances in nanotechnology have led to the development of disease diagnostic and therapeutic agents [1–15]. Nanoparticle-based imaging and therapy using superparamagnetic iron oxide (SPIO), quantum dot, silica, and gold are of interest to the field of theranostic nanomedicine. In particular, SPIO

http://dx.doi.org/10.1016/j.actbio.2016.02.020 1742-7061/Ó 2016 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

Please cite this article in press as: D.H. Nguyen et al., Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.02.020

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D.H. Nguyen et al. / Acta Biomaterialia xxx (2016) xxx–xxx

nanoparticles (NPs) have attracted increasing attention in the fields of cancer imaging, diagnostics, and treatment because of their excellent imagability and their Food and Drug Administration-approved biocompatibility [6]. SPIO NPs possess various desirable features that have been widely applied in magnetic resonance imaging (MRI) [1–6,9,16], drug delivery [1–6,9], and hyperthermia cancer treatments [7]. However, a number of limitations hinder clinical use, including relatively rapid body clearance, low drug loading, and lack of target specificity that ultimately result in insufficient imaging signal intensity or drug concentration at target sites. Many scientists have attempted to address these shortcomings by modifying surface properties, encapsulating SPIOs in micro/nanocarriers, and using internal or external navigation systems [17]. Surface modification, such as the introduction of amphiphilic molecules, bifunctional polymeric ligands, or biomolecules can stabilize SPIO NPs and prevent particle agglomeration. Surface modification of SPIO NPs with polyethylene glycol (PEG), known as PEGylation, has become a common method for inhibiting phagocytosis by the reticuloendothelial system (RES), extending the halflife in blood circulation and promoting the enhanced permeability and retention (EPR) effect in vivo. In addition, SPIO NP size can be increased by clustering them in larger nanocarriers, including nanogels, liposomes, micelles, or capsules, since NP size plays a major role in pharmacokinetics and tissue distribution in vivo. For example, particles 200 nm become concentrated in the spleen or are taken up by phagocytic cells, leading to low plasma concentrations. Interestingly, the encapsulation of SPIO NPs in other carrier systems can also increase magnetic resonance signals. SPIO NP clustering has been found to increase T2 relaxivity and this increase is more significant in larger clusters. To date, many PEGylation methods have been applied for SPIO NPs. For example, PEGs were covalently or ionically linked to iron oxide nanoparticles during particle synthesis using silane coupling agents or a ligand exchange method. However, this process is usually time-consuming and often changes the magnetic properties of SPIO NPs [18]. Amstad et al. identified catechol-derivative anchor groups possessing an irreversible binding affinity to iron oxide; using these anchor groups, ultrastable iron oxide nanoparticles could be obtained [19]. However, such SPIO NPs exhibit low efficacy for drug loading and rapid drug release since the drugs are weakly entrapped by physical adsorption. It is well established that cyclodextrins (CDs) can form inclusion complexes with a wide variety of lipophilic molecules in their hydrophobic cavity, thus improving water solubility, stability, and biological activity in the body [20,21]. Recent studies have attempted to introduce CDs on the surface of SPIO NPs [1,22– 26]; SPIO NPs were prepared by precipitation of iron salts in the presence of ammonia and the particles were mixed with b-CD and a pluronic polymer (F127). However, this method yielded large aggregates or irregular clusters. In this study, a new method for preparing multifunctional selfassembled magnetic nanoclusters (SAMNs) was developed using bCD coated SPIO (SPIO@CD) NPs (Fig. 1). The b-CD was modified with dopamine to enable stable conjugation on SPIO. Surface immobilized CD was utilized to introduce PEG and load paclitaxel (PTX). The terminal PEG was functionalized with a tumor-targeting ligand, cyclo-Arg–Gly–Asp-D-Phe–Cys c(RGDfC), since targeted drug delivery to a specific site through the receptor-mediated endocytosis could improve therapeutic efficiency and minimize side effects. The conjugation of specific ligands (e.g., Arg–Gly– Asp; RGD) is an accepted strategy used to facilitate efficient targeting of drug carriers to tumors since the RGD sequence can recognize molecular signatures on the surface of cancer cells (bio-conjunction) [6,27]. SAMNs were characterized by powder

X-ray diffraction (XRD), Fourier transform infrared spectra (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), vibration sample magnetometer (VSM), and relaxivity measurements. The enhanced cellular uptake of c(RGDfC)-SAMNs was evaluated by flow cytometry and confocal laser scanning microscopy (CLSM). Finally, in vitro MRI visibility was tested. 2. Material and methods 2.1. Materials Superparamagnetic iron oxide nanoparticles (SPIO), bcyclodextrin (CD), 1-adamantylamine (ADA), dopamine hydrochloride (DOPA), poly(ethylene glycol) (PEG, Mw 4000), potassium iodide, silver (I) oxide, p-toluenesulfonyl chloride (TsCl), potassium thioacetate (KSAc), 1,10-phenanthroline, rhodamine B (Rho), N,Ndimethylformamide (DMF), tetrahydrofuran (THF), and stannous 2-ethylhexanoate were obtained from Sigma Aldrich (St Louis, MO, USA). Triethylamine (TEA) and aluminum oxide were purchased from Acros Organics (Morris Plains, NJ, USA). Divinyl sulfone (DVS) and cyclo(Arg–Gly–Asp-D-Phe–Cys) (c(RGDfC)) were supplied by TCI (Tokyo, Japan) and Peptide International (Louisville, KY, USA), respectively. Paclitaxel (PTX) was supplied from Samyang Corporation (Seoul, Korea). All reagents and solvents were used as received without further purification. 2.2. Synthesis of monotosyl-PEG, ADA-PEG, and VS-PEG-ADA Monotosyl-PEG was synthesized using a stoichiometric amount of TsCl in the presence of silver (I) oxide and a catalytic amount of potassium iodide, as previously reported [28]. Briefly, PEG dissolved in MC was added to silver (I) oxide, potassium iodide, and TsCl. Following 2 h of stirring, the solution was filtered and evaporated. The product was obtained by recrystalyzation using MCether co-solvent and the yield was approximately 90%. The degree of substitution was determined to be 93%, calculated based on the integral value of PEG (d 3.67) and the methylene protons adjacent to the tosyl group (d 4.15). 1H NMR (400 MHz, CDCl3, d in ppm): 7.79 (Ar), 7.34 (Ar), 4.15 (CH2OTs), 3.67 (O–(CH2)2–O), 2.75 (CH2–OH), and 2.45 (Ar–CH3) (Fig. S1i). The obtained monotosyl-PEG (8 g, 2.25 mmol) and ADA (0.45 g, 3 mmol) were dissolved in 200 mL of acetonitrile and the mixture was heated at 75 °C for 3 days under nitrogen atmosphere. The solution was allowed to cool to room temperature, dialyzed against water in a dialysis membrane (molecular weight cutoff, MWCO = 3.5 kDa) for 3 days, and subsequently lyophilized. Product yield was approximately 95%. The degree of substitution was determined to be 57%, calculated based on the integral value of PEG (d 3.67) and the methylene protons of the ADA group (d 1.75). 1H NMR analysis results indicated a nearly quantitative transformation of TsCl into ADA moieties. 1H NMR (400 MHz, CDCl3, d in ppm): 3.67 (O–(CH2)2–O), 1.91 (CH–(CH2)3), 1.75 (CH–CH2–CH), 1.50 (CH–CH2–CH) (Fig. S1ii). For the final step of PEG modification, the conjugation of DVS to ADA-PEG was carried out using the following procedure: a mixture of ADA-PEG (4 g, 1 mmol) and potassium tert-butoxide (t-BuOK; 0.56 g, 5 mmol) in 150 mL of MC was slowly dropped into an excess amount of DVS (20-fold) in 30 mL of MC and stirred at 30 °C for 2 days under a nitrogen atmosphere. The solution was then evaporated and precipitated into cold diethyl ether and dried in vacuo. Product yield was approximately 90%. The degree of substitution was determined to be 45%, calculated based on the integral value of PEG (d 3.67) and the proton on the vinyl group (d 6.72). 1H NMR (400 MHz, CDCl3, d in ppm): 6.72 (SO2CH),



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Fig. 1. Schematic illustration of the fabrication of self-assembled magnetic nanoclusters (SAMNs) using b-cyclodextrin-functionalized superparamagnetic iron oxide (SPIO@CD), paclitaxel (PTX), adamantylamine-poly(ethylene glycol)-vinyl sulfone (ADA-PEG-VS), and the cyclo(Arg–Gly–Asp-d-Phe–Cys) (c(RGDfC)) peptide for theranostics.

6.3 – 6.1 (CH–CH2), 3.8 (O–(CH2)2–O), 3.67 (O–(CH2)2–O), 1.91 (CH–(CH2)3), 1.75 (CH–CH2–CH), 1.50 (CH–CH2–CH) (Fig. S1iii). 2.3. Synthesis of 6-TsO-CD and DOPA-CD Mono-6-deoxy-6-(p-tolylsulfonyl)-cyclodextrin (6-TsO-CD) was prepared as previously reported [29]. 1H NMR (400 MHz, DMSO-d6, d in ppm): 7.74 (Ar); 5.7–5.6 (CH–OH); 4.8–4.7 (CH– O); 4.5–4.4 (CH2–O); 3.6–3.4 and 3.6–3.2 (CH–O, overlaps with HOD); and 2.4 (3H) (Fig. S1iv). The conjugation of DOPA to CD (DOPA-CD) was carried out using the following procedure: 6-TsO-CD (5 g, 3.86 mmol) was dissolved in 50 mL of DMF. DOPA (0.88 g, 4.63 mmol) was then added to the solution and stirred for 3 days at 60 °C under nitrogen atmosphere. The solution was evaporated and precipitated into acetone. Following filtration, the final product was obtained by vacuum drying. 1H NMR (400 MHz, DMSO-d6, d in ppm): 6.68 (Ar); 6.48 (Ar); 5.7–5.6 (CH–OH); 4.8–4.7 (CH–O); 4.5–4.4 (CH2–O); 3.6–3.4 and 3.6–3.2 (CH–O, overlaps with HOD); and 2.9–2.7 (CH2) (Fig. S1v). 2.4. Preparation of self-assembled magnetic nanoclusters (SAMNs) 2.4.1. Preparation of CD immobilized SPIO (SPIO@CD) THF/DMF (12.5 mL, 3:2 v/v) containing CD-DOPA (50 mg, 40 lmol) was vortexed for 30 min and sonicated (cycle 3, 20%) for 2 min using a Sonopuls ultrasonic homogenizer (HD 2070, Bandelin, Germany). The solution was added to 25 mL of THF containing SPIO (25 mg) and then vortexed for 30 min and sonicated (cycle 0, 100%) for 2 min. Purified SPIO@CD was deposited and collected using a magnetic bar. 2.4.2. Preparation and functionalization of SAMNs PTX (3 mg, 3.5 lmol) in DMF (10 mL) was mixed with THF (10 mL) containing SPIO@CD (20 mg, 2.6 lmol) and sonicated (cycle 0, 100%) for 1 min. Next, VS-PEG-ADA (100 mg, 25 lmol) was added to the mixture and sonicated (cycle 0, 100%) for 1 min. The solution was then added to 80 mL of deionized water and sonicated (cycle 0, 100%) for 2 min, followed by rotary evaporation at 40 °C for 30 min to remove residual solvent under reduced pressure. Finally, the product was purified by dialysis

against deionized water for 3 days (MWCO 50 kDa) and then lyophilized. To prepare c(RGDfC) functionalized SAMN (c(RGDfC)-SAMN), c (RGDfC) (1.8 mg, 3.12 lmol) was reacted with the VS group of SAMN (30 mg, 2.60 lmol) in 10 mL of phosphate buffer (0.1 M; pH 8) for 6 h [27]. Following this reaction, the mixture was purified by dialysis against deionized water for 1 day (MWCO 50 kDa) and then lyophilized. For further cellular uptake tests, SAMN and c (RGDfC)-SAMN were prepared using Rho/PTX (1:2 wt.%) instead of only PTX. 2.5. Characterization The chemical structure of all products was characterized by 1H NMR (Mercury 400 MHz, Varian Medical Systems Inc., Palo Alto, CA, USA) and FT IR (Magna-IRTM 550 spectrometer, Nicolet, USA). To characterize the SPIO, XRD was performed using a Rigaku DMAX 2000 diffractometer (Rigaku Americas Corporation, Woodlands, TX, USA) equipped with Cu/Ka radiation at a scanning rate of 4°/min in the 2h range of 30–70° (k = 0.15405 nm, 40 kV, 40 mA). TGA was conducted to determine the amount of SAMN component using a Q50 TGA (TA Instruments, New Castle, DE, USA) from 23 °C to 600 °C with a heating ramp of 10 °C, under a constant flow (100 mL/min) of argon. The iron levels in the formulation were determined using the 1,10-phenanthroline colorimetric method [30]. Absorbance was measured at 511 nm using a spectrophotometer (Jasco V-570; Jasco Inc., Easton, MD, USA). Particle size and size distribution were determined using the Zetasizer Nano ZS (ZEN 3600, Malvern Instruments, Malvern, UK) at 25 °C. SPIO and SPIO@CD particles were prepared in THF:H2O (2:1, v:v) and SAMN and c(RGDfC)-SAMN were prepared in H2O at a concentration of 1 mg/mL. All samples were filtered using a syringe filter (pore size of 0.45 lm) and sonicated for 10 min prior to measurement [31]. SAMN morphology and size were imaged by TEM using a JEM-3000F microscope (JEOL, Tokyo, Japan). The samples were prepared by placing a drop of solution onto a carbon–copper grid (300-mesh; Ted Pella, Inc., Redding, CA, USA) and air-drying for 10 min. The SPIO and SAMN magnetization data were determined using a superconducting quantum interference device (SQUID) magnetometer at 300 K. The applied magnetic field was varied


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from 4 104 Oe to 4 104 Oe in order to generate hysteresis loops. The magnetic responsiveness of SPIO and SAMN was tested by placing a magnet near the glass vial. 2.6. PTX loading contents and in vitro PTX release The SAMN PTX loading contents was determined by high performance liquid chromatography (HPLC; Prominence LC-20A, Shimadzu, Kyoto, Japan). PTX loaded SAMN was removed from the solution using a magnetic bar during the SAMN preparation step, prior to evaporation. The remaining solution was lyophilized and dissolved in acetonitrile/water solution (60:40, v:v). A 10 ll aliquot of the sample was injected and the mobile phase was delivered at a flow rate of 1.00 mL/min. A reverse phase Fortis C18 column (150 4.6 mm i.d., pore size 5 lm, Fortis Technologies Ltd., Cheshire, UK) was used. Column effluent was detected at 227 nm with a UV detector. The calibration curve for PTX quantification was linear over the standard PTX concentration range (0–20,000 ng/mL) with a correlation coefficient of R2 = 0.994. The following equations were used to calculate the drug loading efficiency (DLE) and encapsulation efficiency (DEE):

DLEð%Þ ¼ ½weight of fed drug weight of unloaded drug =weight of SAMN 100% DEEð%Þ ¼ ½weight of fed drug weight of unloaded drug =weight of fed drug 100% In vitro PTX release experiments were performed in PBS buffer (0.01 M, pH 7.4; containing 0.5 wt% Tween-80) at 37 °C using a dialysis method [32]. One milliliter of SAMN suspended in PBS (PTX content, 0.3 mg/mL) was transferred to a dialysis bag (MWCO = 12–14 kDa) and immersed into 14 mL of medium at 37 °C. The vials were then placed in an orbital shaker bath maintained at 37 °C and shaken horizontally at 100 rpm. At specific time intervals, 14 mL of the released medium was collected and an equal volume of fresh medium was added. Following lyophilization of the collected medium, the amount of released PTX was determined using HPLC. 2.7. Cellular uptake study HeLa cells were purchased from the American Type Culture Collection. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. The cellular uptake behavior and the intracellular distribution of the SAMNs were analyzed using both fluorescence-activated cell-sorting (FACS) and confocal laser scanning microscopy (CLSM). For FACS analysis, HeLa cells were seeded onto a 6-wellplate (5 106 cells/well) and cultured overnight. The cells were treated with SAMNs or c(RGDfC)-SAMNs for 4 h (SAMN concentration: 50 lg/mL). The treated cells were then washed 3 times with PBS, trypsinized at 37 °C, centrifuged (1500 rpm, 5 min), and resuspended in PBS (pH 7.4, 1% BSA). Finally, fluorescence histograms were obtained using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using the CellQuestTM software supplied by the manufacturer. Each histogram was generated by analyzing at least 10,000 cells. For CLSM studies, HeLa cells were seeded on a coverslip in a 24well plate (5 104 cells/well) and cultured overnight to allow cell attachment. The cells were treated with SAMNs and c(RGDfC)SAMNs for 4 h (SAMN concentration 50 lg/mL), washed 3 times with PBS, and fixed with paraformaldehyde for 10 min. The cells were then washed 3 times with PBS, treated with 40 ,6-diamidino-

2-phenylindole (DAPI) for 15 min for nuclei staining, and washed 3 times with PBS. Finally, the cells were mounted in VECTASHIELDÒ anti-fade mounting medium (Vector Labs, Burlingame, CA, USA) and imaged using a confocal laser scanning microscope (CLSM; Zeiss LSM 510; Carl Zeiss, Oberkochen, Germany). Images were analyzed using image software (Carl Zeiss LSM). Rho and DAPI were visualized in red and blue, respectively. 2.8. Cytotoxicity assay HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillinstreptomycin at 37 °C under a humidified atmosphere containing 5% CO2. The cells were seeded into a 96-well plate (1 104 cells/ well) and incubated overnight at 37 °C. The media was then replaced with a suspension of samples at various concentrations in 2% (v/v) of tween 80, followed by incubation for 48 h. Next, 25 lL of MTT solution (2 mg/mL in PBS, pH 7.4) was added to the wells, and the cells were further incubated for 3 h. The media was removed, formazan crystals were dissolved in DMSO (130 lL/well), and the absorbance was obtained at 570 nm using a microplate reader (SpectraMax M2e, Molecular Devices Co., USA). 2.9. Relaxivity measurements To verify the contrast characteristics of SAMN as an MRI contrast agent, the transverse (1/T2) relaxation rate was calculated based on the MRI. Synthesized c(RGDfC)-SAMN and SAMN were compared with SPIO@CD and SPIO at various iron concentrations. SAMN and c(RGDfC)-SAMN were prepared in deionized water while SPIO@CD and SPIO were prepared in deionized water containing Tween 80 (10 wt%) to increase their solubility. All MR relaxivity measurements were performed with a 1.5 T clinical MRI instrument (GE Signa Excite Twin-Speed, GE Healthcare, Milwaukee, WI, USA) using a knee coil. A T2-weighted conventional spin echo sequence was acquired with a repetition time (TR) of 2400 ms; various echo time (TE) points, including 12, 16, 20, 24, 32, 36, 40, 48, 60, 64, 80, 120, 160, 180, and 240 ms; 14 cm field of view; slice thickness of 2 mm; interslice gap of 0.1 mm; and 256 192 matrices. The specific r2 relaxivity values were calculated by curve fitting 1/T2 (s1) versus the iron (Fe) concentration (mM). 2.10. Statistical analysis All experimental data were analyzed using Student’s t test. P < 0.05 was considered to be statistically significant. The results are represented as the mean ± standard deviation. 3. Results and discussion 3.1. Characterization of SAMNs PTX is an effective drug for treating a broad range of solid tumors, including ovarian cancer, non-small cell lung cancer, and breast cancer. PTX can be hosted by b-CD on SPIO NPs; PTXloaded multifunctional SAMNs were fabricated by self-assembly. The DLE and DEE of PTX in SAMNs ranged from 4.9% to 11.4% and 42.5% to 77.8%, respectively, depending on SAMN composition (Table 1). The ADA group on PEG was also complexed with b-CDs to PEGylate the SPIO NPs, stabilizing SPIO nanoclusters in the aqueous phase. ADA is known as one of the guest molecules of b-CD [33]. b-CD was chemically modified with DOPA that was used to conjugate b-CDs on SPIO NPs. The binding of DOPA-CD and VS-PEG-ADA components with SPIO was confirmed by FT IR


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D.H. Nguyen et al. / Acta Biomaterialia xxx (2016) xxx–xxx Table 1 Physical characteristics of nanoparticles. Particle cord

PEG (%)a

Iron oxide (%)b

PTX loading DEE (%)

SPIO SPIO@CD SAMN (PEG:SPIO = 0:1) SAMN (PEG:SPIO = 1:1) SAMN (PEG:SPIO = 3:1) SAMN (PEG:SPIO = 5:1) c(RGDfC)-SAMN (PEG:SPIO = 5:1) a b c d e f g

– – 0 46.5 72.3 81.3 81.3

73.4 ± 3.0 76.3 ± 3.0 72.4 ± 2.6 65.2 ± 3.1 60.1 ± 2.4 56.1 ± 2.1 57.6 ± 2.7

c

– – 77.80 ± 0.72 78.15 ± 0.42 77.17 ± 1.40 52.77 ± 1.36 42.50 ± 4.06

Diameter DLE (%)

d

– – 11.44 ± 0.11 9.16 ± 0.05 9.34 ± 0.17 5.82 ± 0.15 4.90 ± 0.46

Size (nm)e

Z-Ave (nm)f

PDIg

7.9 ± 1.1 8.3 ± 1.1 – – – 80.4 ± 8.4 87.1 ± 10.2

8.63 10.51 274.1 203.6 133.1 91.7 116.3

0.303 0.165 0.313 0.278 0.256 0.170 0.190

The weight percentage of PEG added during the preparation of SAMN. Iron oxide level was calculated as the weight percentage of iron oxide divided by the total particle weight. Drug encapsulation efficiency (DEE) was calculated as the percentage of loaded PTX divided by the original feeding amount. Drug loading efficiency (DLE) was calculated as the weight percentage of PTX divided by the total particle weight. Size was measured by TEM. Hydrodynamic diameter was measured by DLS. The polydispersity index (PDI) was measured by dynamic light scattering.

spectroscopy (Fig. S2). The results clearly demonstrate that the characteristic absorption band of the oleic acid ligands (stretching vibration –CH2–) on the surface of bare magnetic nanoparticles was 2877 cm1. In the SPIO@CD spectra, the appearance of characteristic peaks of 1027 cm1 (stretching vibration of O–H), 1630 cm1 (stretching vibration of N–H), 2877 cm1 (stretching vibration of C–H), and the broad band at 3300–3450 cm1 (stretching vibration of –OH) indicated DOPA-CD deposition on the SPIO. The appearance of characteristic peaks of 1247 cm1 (stretching

vibration of C–O–C) and 2785 cm1 (stretching vibration of C–H) indicated VS-PEG-ADA deposition on the SPIO@CD. The composition and hydrodynamic diameter of SPIO, SPIO@CD, and SAMNs are listed in Table 1. SPIO size (8 nm) slightly increased when coated with DOPA-CDs (10 nm). CD was used to conjugate PEG in order to form stable SAMNs with a tunable size ranging from 90 to 270 nm. A smaller and narrower SAMN size distribution was obtained as more PEG was used for formulation, indicating that PEG stabilized the nanoclusters. As demonstrated in

Fig. 2. Correlation between size and composition of SAMNs: (a) weight% of PEG in feed (y = 2.155x + 283.43, R2 = 0.9583); and (b) weight% of SPIO in SAMNs (y = 11.385x 546.74, R2 = 0.995). The composition and size of SAMNs were determined by TGA and Zetasizer Nano ZS, respectively. (c) TEM images for (i) SPIO NPs; (ii) SPIO@CD; (iii) SAMNs, and (iv) c(RGDfC)-SAMNs.


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Fig. 2a and b, size exhibits a linear relationship with the weight ratio of PEG and SPIO (R2 > 0.95). Size tunability is highly advantageous since body-residence time and targetability to specific organs/cells can be controlled by different particle sizes. Particle size determines blood circulation time, organ distribution, cell uptake kinetics, and body clearance time. For instance, particles with sizes 200 nm become concentrated in the spleen. Particles with a size range of 10–200 nm are considered to be optimal for longer circulation times since they can evade RES uptake. The morphology of SPIO, SPIO@CD, and SAMN were elucidated by TEM. Fig. 2c-i and c-ii show representative images of prepared SPIO and SPIO@CD NPs. The mean diameter of SPIO NPs and SPIO@CD was approximately 9 nm and 10 nm, respectively, which is consistent with the results obtained from DLS. SPIO and SPIO@CD were well dispersed and uniform in shape and size, however, agglomerates existed due to the absence of surface stabilizers. In contrast, the core-shell structure of the magnetic nanoclusters shown in Fig. 2c-iii and c-iv did not reveal any aggregates. SAMN composition was analyzed by TGA (Fig. 3a). Loss of oleic acid in SPIO NPs was approximately 17.7% in weight at 230–600 °C (Fig. 3a). At this temperature range, the weight loss of the SPIO@CD and SAMNs was 16.4% and 34.6%, respectively, mostly due to thermal degradation of DOPA-CD, VS-PEG-ADA, and PTX. A relatively high weight% of SPIO (57.6–72.4%) was incorporated in the SAMNs. Fig. 3b compares the XRD-diffraction patterns of SPIO and SAMNs;

the XRD patterns exhibit six characteristic peaks, indicating cubic iron oxide crystals (2h = 30.08, 35.38, 42.84, 53.34, 56.92, and 62.42), which are close to the JCPDS for magnetite (card No. 893854). These are related to their corresponding indices (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0), respectively [34]. A large decrease in saturation magnetization (Ms) is undesirable since it will prevent high imaging signals at the target site. The most effective method for enhancing the Ms. of SPIO is to increase particle size. However, a synthetic method for producing high-quality particles >20 nm has yet to be developed. Moreover particles >20 nm will become non-superparamagnetic. As shown in Fig. 3c, one way to enhance the Ms. is clustering the SPIO NPs; the Ms of the SPIO NPs increased from 34.5 to 45.0 emu/g. According to Poselt et al., the Ms of SPIO NPs could be tuned by controlling cluster size [35]. There was no apparent hysteresis or remanence in the hysteresis loop and the coercivity was zero, indicating that SPIO superparamagnetism was retained in the SAMNs. Fig. 3d shows that the SAMNs could be easily separated from the solution within a few seconds. 3.2. Controlled release of PTX Spatially and temporally controlled delivery of anticancer drugs is required for improving drug efficacy and reducing side effects. SPIO NPs have typically been used to modulate drug release with an external magnetic force. It has been well established that SPIO NPs vibrate under a magnetic field and that the vibration acceler-

Fig. 3. In vitro characterization: (a) TGA curves of SPIO (square dot), SPIO@CD (round dot), and SAMNs (solid line); (b) XRD patterns of SPIO and SAMNs; (c) magnetization curve of SIPO (s) and SAMNs (d) at 298 K measured by SQUID exhibiting magnetic saturation; (d) image of the magnetic separation of SAMNs using a magnet.



ates drug release from the NPs [36]. However, in the absence of a magnetic stimulus, it might prove difficult to control the SPIO NP drug release kinetics. Recently, Kim et al. demonstrated that the addition of competitive molecules together with the drug can trigger drug release from carriers inside living cells [37]. In this study, the introduction of a cyclic compound on the Au particle surface diminished cytotoxicity; the cyclic compound was then removed by the administration of a competitive molecule (ADA), greatly increasing cytotoxicity in the cells.

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PTX release triggered by the presence of competitive guest molecules was studied in PBS at 37 °C. Free ADA was used as a model competitor of PTX because adamantyl groups possess higher affinity to b-CD than PTX [38,39]. As shown in Fig. 4a, only 9.6% PTX was released over 120 h. The host-guest interactions between PTX and CD could effectively preserve the structural integrity of SAMNs [13], which might lead to a slower drug release rate. In contrast, in the presence of competitor molecules, the PTX release rate from the SAMN significantly increased up to 44.1% over the same

Fig. 4. (a) In vitro PTX release profiles from SAMNs (d) with and (s) without a complex competitive molecule (ADA) of b-CD. (b, c) Cellular uptake of SAMNs in HeLa cells. (b) Flow cytometry analysis and (c) CLSM images of HeLa cells treated with (i) PBS, (ii) SAMNs, and (iii) c(RGDfC)-SAMNs at 37 °C for 4 h. (d, e) Dose-dependent cytotoxicity of (d) c(RGDfC)-SAMN without PTX (D). The percentage of cell viability of HeLa cells when treated with (e) free PTX (◆) and PTX-containing c(RGDfC)-SAMN (d) after 48 h of incubation (n = 4) (⁄⁄⁄⁄P < 0.0001).


8


Fig. 5. Magnetic resonance characteristics of magnetic nanoparticle formulations: (a) T2 relaxation rate (1/T2) and (b) signal intensity T2-weight MR images of aqueous solutions containing (i) SPIO NPs; (ii) SPIO@CD; (iii) SAMNs, and (iv) c(RGDfC)-SAMNs with different Fe concentrations.

time period. This result demonstrates that most of the PTX was loaded by complexation with b-CD and non-specific loading of PTX was minimal; thus, following intracellular uptake of the SAMNs, PTX could be released by the destabilization of the clusters.

3.3. Cellular uptake of SAMNs Rho was hosted by CDs as a fluorescence marker [40]. Flow cytometry and CLSM were used to evaluate the effect of c(RGDfC) on the cellular uptake of SAMNs in HeLa cells. Fig. 4b shows flow cytometry histograms based on Rho fluorescence from HeLa cells incubated with SAMNs or c(RGDfC)-SAMNs for 4 h. Cells without any treatment were used as a the negative control and showed only low-level autofluorescence. c(RGDfC)-SAMN-treated HeLa cells showed higher fluorescence levels compared to those treated with bare SAMNs. This result indicates that c(RGDfC) accelerated the cellular uptake of SAMNs subsequent to efficient conjugation with PEGs and b-CDs. Enhanced uptake was due to an unusual route of endocytosis that is mediated by the integrin avb3 receptor on the cell surface [27,41]. CLSM was used to visualize the nanoclusters in the cells in order to determine the intracellular distribution. As predicted, brighter fluorescence was observed in HeLa cells incubated with c(RGDfC)-SAMNs compared with SAMNs incubated without the peptide (Fig. 4c). In both cases, the nanoclusters appeared in the cytoplasm and at much lower levels in the nucleus, indicating that uptake occurred via a receptor-mediated endocytosis process. These SAMNs could be utilized in eliminating micrometastasis tumors since the c(RGDfC) peptide is highly specific to metastatic tumor cells.

3.4. Cytotoxicity studies Fig. 4d shows the dose-dependent cytotoxicity of c(RGDfC)SAMN without PTX. Over 80% of cells were viable after incubating for 2 d even at the concentration of the nanoclusters as high as 250 lg/mL. The cytotoxicity of c(RGDfC)-SAMNs containing PTX was evaluated in Fig. 4e. The viability of HeLa cells treated with the SAMNs was significantly reduced in a manner corresponding to the concentration of PTX in the clusters, and only 56% of cells were viable at 20 lg/mL of PTX. The equivalent amount of free PTX led to a more dramatic decrease in the cell viability due to

the instant exposure of the PTX to the cells, which has been previously reported in the literature [42]. 3.5. Relaxivity measurement SPIO and modified SPIO products have been used clinically as T2-type MR negative contrast agents. As previously reported [43,44], assembled or aggregated SPIO NPs can significantly enhance T2 relaxivity (r2) due to synergistic magnetism. Here, the proton transverse relaxivity (r2) of SPIO NPs, SPIO@CD, SAMNs, and c(RGDfC)-SAMNs were measured. The data in Fig. 5 indicate a linear relationship between the water proton transverse relaxation rate (1/T2) and Fe concentration. The specific relaxivity values (r2) of SPIO and SPIO@CD were 199.24 and 161.38 s1 mM1 Fe, respectively. However, the T2 relaxivity (r2) of c(RGDfC)-SAMNs and SAMNs increased up to 265.05 and 261.56 s1 mM1 Fe, respectively (33% enhancement). This difference in enhancement can be attributed to cluster size and density. Larger and more condensed SPIO clusters exhibited higher transverse relaxivity [45]. 4. Conclusions Theranostics, defined as disease treatment combining diagnostic and therapeutic capabilities within a single system, has become a rational therapeutic paradigm for monitoring disease statues prior to, during, and post, treatment. In this study, selfassembled magnetic nanoclusters were prepared as a multimodal theranostic agent by complexation of both a tumor-targeting moiety (c(RGDfC)-conjugated polyethylene glycol) and an anti-tumor drug (paclitaxel) with cyclodextrins covalently immobilized on the surface of iron oxide nanoparticles. The advantages of magnetic nanoclusters include tunable cluster size, efficient drug loading, increased T2 relaxivity, and enhanced particle uptake in cancer cells with no apparent decrease in the inherent magnetization characteristics. Given these versatile features, magnetic nanoclusters should be highlighted as one of the most promising platforms for the next stage modality of cancer treatments. Acknowledgements This work was supported by National Reaserch Foundation (NRF) grant funded by the Korean government, MSIP (NRF2015M3A9E2028578 and NRF-2015R1A2A1A14027221).



Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2016.02. 020.

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