Gold Ion Nanoprobes for MR Imaging and

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Theranostic Iron-oxide/gold-ion nanoprobes for MR imaging and non-invasive RF hyperthermia Sajid Fazal, Bindhu Paul-Prasanth, Shantikumar Nair, and Deepthy Menon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08939 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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ACS Applied Materials & Interfaces

Theranostic Iron-oxide/gold-ion nanoprobes for MR imaging and non-invasive RF hyperthermia Sajid Fazal, Bindhu Paul-Prasanth, Shantikumar V. Nair*, Deepthy Menon*

Amrita Centre for Nanosciences and Molecular Medicine, Amrita University, Kochi-682041, Kerala India Fax: +91 484 2802030; Tel: +91 484 4008750; *E-mail: [email protected], [email protected] Keywords: radiofrequency ablation, iron-oxide nanoparticles, magnetic resonance imaging, cancer, theranostics

ABSTRACT This work focuses on the development of a nanoparticulate system that can be used for MR imaging and E-field non-invasive radiofrequency (RF) hyperthermia. For this purpose, an amine-functional gold ion complex, (GIC) [Au(III)(diethylenetriamine)Cl]Cl2 which generates heat upon RF exposure, was conjugated to carboxyl functional polyacrylic acid capped iron-oxide nanoparticles (IO-PAA NPs) to form IO-GIC NPs of size ~100 nm. The multimodal superparamagnetic IO-GIC NPs produced T2-contrast on MR imaging and unlike IO-PAA NPs, generated heat on RF exposure. The RF heating response of IO-GIC NPs was found to be dependent on RF power, exposure period and particle concentration. IO-GIC NPs at a concentration of 2.5 mg/mL showed a high heating response (δT) of ~40°C when exposed to 100W RF power for 1 minute. In-vitro cytotoxicity measurements on NIH-3T3 fibroblast cells and 4T1 cancer cells showed that IO-GIC NPs are cytocompatible at high NP concentrations for up to 72 hours. Upon in-vitro RF exposure (100W, 1 minute), a high thermal response lead to cell death of 4T1 cancer cells incubated with IO-GIC NPs (1mg/mL). 1 ACS Paragon Plus Environment


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H&E imaging of rat liver tissues injected with 100 µL of 2.5 mg/mL IO-GIC NPs and exposed to low RF power of 20W for 10 minutes showed significant loss of tissue morphology at the site of injection, as against RF exposed or nanoparticle injected controls. In-vivo MR imaging and non-invasive RF exposure of 4T1 tumor bearing mice after IO-GIC NP administration showed T2 contrast enhancement and a localized generation of high temperatures in tumors leading to tumor tissue damage. Further, the administration of IO-GIC NPs followed by RF exposure showed no adverse acute toxicity affects in-vivo. Thus, IO-GIC NPs show good promise as a theranostic agent for magnetic resonance imaging and noninvasive RF hyperthermia for cancer.

Introduction Cancer, being a highly heterogeneous disease, has eluded “the one-size fits all” paradigm of modern medicine.1 This begat the idea of personalized medication for cancer, wherein the choice of drug, its dosage and schedule are decided based on the tumor characteristics such as tumor size, grade and biology.2 One approach towards such personalized medication is to systematically plan and design the course of therapy based on real-time assessment of tumor characteristics using appropriate therapeutic and diagnostic agents.3 However, individually administered diagnostic and therapeutic agents, would show different biodistribution and clearance pattern,4 which would hamper treatment efficiency. To counter this, the concept of theranostics emerged, wherein both therapeutic and diagnostic modalities are combined together in a single platform so as to maximize therapeutic outcomes.3-4 Nanomaterials, owing to their diverse properties and simple synthesis chemistries, have been extensively investigated in the recent past for such theranostic applications.5 The different modalities that have been combined to demonstrate theranostic ability using nanomaterials include, MR imaging and photodynamic therapy,6 CT imaging and drug delivery,7 PET imaging and

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photothermal therapy,8 optical imaging and magnetic hyperthermia9 and similar other combinations.10-11 Radiofrequency ablation (RFA) therapy is a modality which is currently used for the treatment of cancers of the liver and breast,12-13 wherein radiofrequency waves deliver lethal thermal doses to tumor sites in an invasive manner using probes. However, this therapeutic modality is limited by the size and location of the tumor.14 In this regard, non-invasive RFA therapy, which does not involve the use of probes, is emerging as a novel therapeutic technique.15 This could be used for the treatment of any deep-seated tumors in the body, in a non-contact fashion due to the low absorbance coefficient of RF waves in body tissues.16 It is noted that RF absorbing materials if present in the vicinity of tumor can enhance its therapeutic response.17 Incidentally, numerous reports have investigated the thermal response of nanoparticles such as gold18 and silica,19 carbon nanotubes (CNT)20, C6021 and quantum dots22 under radiofrequency fields for non-invasive RFA of tumor. Concurrently, a number of theoretical reports have tried to elucidate the mechanism of interaction between nanoparticles and radiofrequency waves and the phenomenon behind the observed heating effects. Reports suggest that CNTs23 have the ability to produce thermal effects due to the contribution of its dielectric loss to the medium. On the other hand, unaggregated gold nanoparticles of size ~ 5 nm produce RF heating effects under high electric field strength (~90 Kv/m) due to the electrophoretic motion of charged particles over finite distances of Å length scales.24 Even though the exact mechanism of interaction between RF waves and nanoparticles have not yet been clearly understood,25 there is a general consensus that ions either in the medium or as stabilizers on nanoparticle surface are important for E-field RF heating activity.25-27 For clinical applicability of non-invasive RFA therapy using nanoparticles, it is vital to increase nanoparticle mediated thermal response, while simultaneously decrease the interaction of RF with body tissues.28 This can be done by engineering nanoparticles that dissipate high thermal energy over short time periods upon RF wave exposure within the 3 ACS Paragon Plus Environment


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tumor vicinity. However, it was recently reported that RF heating is dependent on sample holder geometry16, 28 and the same sample can produce markedly different heating effects when the sample holder orientation and shape varied under a uniform E-field. This implies that, RF mediated thermal response can also be modulated by varying the volume of interaction of the nanoparticles and the RF field. Thus, by engineering imageable nanoparticles that also produce heat on RF exposure, nanoparticle-RF interaction can be maximized by aligning RF wave exposure according to the spatial distribution of the nanoparticles in the tumor. With this aim of generating better thermal response and thereby improved therapeutic outcome, we report for the first time the development of iron-oxide nanoparticles that exhibit both E-field non-invasive RF hyperthermia and MR imaging capabilities. In this study, we probe the RF heating effects of a nanoparticulate system viz; IO-GIC NPs comprised of MR imageable iron-oxide nanoparticles coupled with an ionic gold complex, GIC, in a capacitatively coupled 13.56 MHz RF generator. The in-vitro and in-vivo capability of this multicomponent system for cancer theranostic applications is demonstrated. Results and Discussion

Design of Iron-Oxide-Gold Ion Complex Nanoparticles (IO-GIC NPs) The primary objective of this study was to design a nanoparticulate system that would incorporate materials for both MRI diagnosis and RF hyperthermia. For this, iron oxide nanoparticles, a well know T2 contrast MR imageable agent, was conjugated to an ioniccomplex of gold viz; [Au(III)(diethylenetriamine)Cl]Cl2) - a material which generates heat on RF exposure. To enable conjugation, iron-oxide nanoparticles were carboxyl functionalized to interact with the amine functional groups of the gold ion complex (GIC) as described in Scheme 1. First, hydrophobic oleic acid capped IO-OA NPs were synthesized29 by the thermal decomposition of Iron-oleate complex at 320ºC (Scheme 1 A). The trioctylphosphine oxide 4 ACS Paragon Plus Environment

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capping was then ligand exchanged with polyacrylic acid, a carboxyl rich biocompatible stabilizing agent30-33 to generate hydrophilic polyacrylic acid capped IO-PAA NPs (Scheme 1 C i). GIC was synthesized separately by reacting ice-cold diethylenetriamine with chlorauric acid

dissolved

in

HCl

(Scheme

1

B)

to

yield

a

yellow-orange

precipitate

[Au(III)(diethylenetriamine)Cl]Cl2 which was washed with diethyl ether and separated.34-35 The free carboxylic acid groups of IO-PAA were then conjugated to the amine functional group of GIC using EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)NHS(N-hydroxysuccinimide) coupling chemistry, to form the theranostic nanoparticle IOGIC NPs (Scheme 1 C ii). An evident color change was also observed after the conjugation of IO-PAA NPs to GIC (Figure S1) wherein yellow colored IO-PAA NPs turned to dark grey.

Scheme 1. Synthesis of IO-GIC NPs from IO-PAA NPs. (A) Synthesis of IO-OA NPs from Iron-Oleate complex by thermal decomposition at 320 °C. (B) Synthesis of GIC from HAuCl4. (C i) Ligand exchange of IO-OA NPs with PAA to form IO-PAA NPs. (C ii) EDC/NHS



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coupling chemistry is used to conjugate the amine of GIC to the carboxyl of IO-PAA, yielding IO-GIC NPs.

Characterization of IO-GIC NPs IO-OA, IO-PAA and IO-GIC NPs were characterized using DLS and zeta potential measurements to understand the effects of ligand exchange and conjugation on its hydrodynamic diameter and charge. IO-OA NPs have a small core diameter (~ 12 nm) and net neutral surface charge (Figure S2) as reported earlier for oleic capped iron-oxide nanoparticles synthesized at 320°C.36 In comparison, IO-PAA and IO-GIC NPs have higher hydrodynamic diameters of 57±22.57 and 110±22 nm respectively (Figure 1 A & B) possibly due to the steric hindrance offered by the ligands, viz., PAA and GIC, present on the core iron oxide nanoparticles. However, the core size of IO-PAA and IO-GIC NPs from TEM analysis was measured to be ~12 nm (Figure 1 E & F), similar to that of hydrophobic IO NPs. The HRTEM images of IO-OA, IO-PAA and IO-GIC NPs further show that the particles are small sized and highly crystalline in nature as evident from the observed well ordered crystallographic planes (Figure S3). Additionally, the surface charge of IO-PAA NPs increased from -33 to -21 mV upon conjugation with GIC (Figure 1 C & D), which can be attributed to the presence of positively charged GIC molecules.


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Figure 1. Hydrodynamic diameters of (A) IO-PAA and (B) IO-GIC NPs. Zeta potential values of (C) IO-PAA and (D) IO-GIC NPs. (E) and (F) show the low and high magnification TEM images of IO-PAA and IO-GIC NPs respectively depicting no significant changes in their size.

X-ray photoelectron spectroscopy (XPS) was done to study the surface chemical composition of IO-GIC NPs (Figure 2). The split photoemission peaks of Au3+ in the IO-GIC XPS spectrum arising from the spin-orbit coupling effects after deconvolution can be seen at 86.4 eV and 87.7 eV for Au 4f7/2 orbital and 89.6 and 92.2 eV for Au 4f5/2 orbital respectively (Figure 2 A-i). Similar spectral positions corresponding to Au3+ have been reported in literature.37-38 The N 1s photoemission band in IO-GIC at 399 eV is also clearly visible in Figure 2 A-ii. These spectra of Au 4f and N 1s bands were also visible in GIC (Figure S4) which confirms the presence of GIC containing Au3+ and NH2 functional groups on the synthesized IO-GIC NPs. Additionally, the split photoemission peaks of Fe 2p at 2p1/2 and



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2p3/2 at 712 and 726 eV respectively confirmed the presence of iron its various oxidation states in IO-GIC NPs (Figure 2 A-iii).

Figure 2. (A) XPS spectra of IO-GIC NPs showing the photoemission peaks of (i) Au 4f7/2 and 4f5/2 orbitals (ii) N 1s orbital and (iii) Fe 2p1/2 and 2p3/2 orbitals confirming the presence of Au3+ and Fe in IO-GIC NPs. (B) Comparative FTIR spectra of IO-PAA & IO-GIC NPs with GIC. The presence of 1645 cm-1 amide 1 band confirms conjugation. (C) VSM analysis of IO- PAA and IO-GIC NPs depicting no hysteresis. (D) T2 imaging of IO-PAA and IO-GIC NPs in 1% agar solution depicting darker contrast with increasing NP concentration. (E) Calculation of transverse relaxivity (R2) from the slopes of the plot of 1/T2 (sec-1) against Fe concentration (mM) FTIR spectral analysis was done to study the chemical changes brought about by the conjugation of GIC to IO-PAA as shown in Figure 2 B. The most prominent peaks of IOPAA lie at 1710 cm-1 which correspond to the highly abundant –COOH functional group of PAA, and at 2850 cm-1 and 2920 cm-1 corresponding to the alkyl –CH stretch, originating from the backbone of the remnant oleic acid chains on the nanoparticle left over after ligand exchange.29, 39 For GIC, the peak at 1600 cm-1 corresponds to its –NH2 scissor vibrations, while the peak at 3412 cm-1 corresponds to the –NH2 stretching vibrations. For IO-GIC, an amide 1 band evident at 1645 cm-1 confirms the conjugation of IO-PAA NPs to GIC molecule. 8 ACS Paragon Plus Environment

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Further, both IO-PAA and IO-GIC NPs show the characteristic Fe-O stretch band at 574 cm1

.The presence of gold in IO-GIC NPs was confirmed by ICP elemental analysis which

showed that ~ 5µg of Au was present in every 1000 µg of IO-GIC NPs.

To determine the ability of the nanoparticles to be used as a magnetic contrast agent, VSM and relaxivity measurements were carried out. Figure 2 C depicts the change in magnetization of the nanoparticles with varying magnetic field strengths. It can be seen that IO-PAA and IOGIC showed a saturation magnetization value of 4.68 and 4.38 emu/g respectively. As expected, the addition of GIC to IO-PAA has led to a small reduction in its saturation magnetization value due to the reduction in the iron content per unit mass of the nanoparticle. (The %mass of Fe in IO-PAA and IO-GIC NPs was ~ 22.1 and 8.3% respectively from ICP analysis). This low value of saturation magnetization might be due to the relatively high concentration of PAA present on IO NPs. However, it is important to mention that the MR contrast offered by the material was not severely affected due to this. Nevertheless, the presence of zero hysteresis confirms the superparamagnetic nature of the synthesized nanoparticles. To study the ability of iron-oxide containing nanoparticles to produce T2-contrast in MRI applications, relaxivity measurements of IO-PAA and IO-GIC NPs were carried out at different concentrations. The reduction in T2 relaxation time and the subsequent increase in dark contrast are represented in Figure 2 D. It can be seen that with increasing nanoparticle concentration, there is a subsequent reduction in T2 relaxation time and increase in darker contrast, clearly implying that these nanoparticles have the ability to produce contrast in body tissues. The relaxivity values obtained from the plot of 1/T2 vs Fe concentration depicted in Figure 2 E for IO-PAA and IO-GIC NPs is 117.21 and 162.71 mM-1sec-1 respectively. As obvious, the relaxivity of IO-GIC is higher than that of IO-PAA, perhaps owing to the



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paramagnetic behavior of gold ions (Au3+) containing two lone pair of electrons, as against that of metallic gold which is diamagnetic.

RF heating activity of IO-GIC NPs The RF heating properties of the synthesized material were studied in a capacitively coupled apparatus developed in-house which was connected to a 13.56 MHz RF generator. Figure S5 shows the RF generator which is connected to the RF shielded cabinet housing the capacitor plates, within which the samples were placed for RF heating. Before evaluating the RF heating of the synthesized nanoparticles, the thermal response of GIC, gold chloride and gold nanoparticles at a fixed concentration (250 µg/mL) was evaluated against PBS buffer, 0.9% NaCl, and milli-Q water as controls. From figure S6 A it can be seen that ionic solutions of gold viz, HAuCl4 and GIC, showed a very high thermal response, while metallic gold in the form of gold nanoparticle exhibited a thermal response similar to that of milli-Q water at 100W RF power for 1 minute exposure. The presence of gold ions in an aqueous medium alters the conductivity of the solution, thereby leading to a positive thermal response on RF exposure. Further, PBS buffer and 0.9% NaCl solutions showed low thermal response on RF exposure. The RF heating response of GIC measured as a function of concentration (Figure S6 B) exhibited a bell shaped heating profile with maximum heating shown at 62.5µg/mL. This bell shaped heating profile is similar to that of NaCl solution reported elsewhere.43 This can be attributed to the variations in electric field strength brought about by the changing dielectric value of the medium. The RF heating effects of the final nanoconjugate, i.e., IO-GIC NPs was tested with saline, milli-Q water and IO-PAA NPs as controls after magnetically separating and washing the particles twice with milli-Q water. Figure 3 A depicts that 1 mg/mL of IO-GIC showed the highest heating response (20 ± 3.5 °C) compared to IO-PAA and 0.9% saline at 100 W RF power for an exposure period of 1 minute. This implies that by conjugating GIC with IO-PAA, 10 ACS Paragon Plus Environment

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RF heating functionality is imparted to IO-PAA NPs, which otherwise by itself showed an RF heating response equivalent to milli-Q water. To negate any inter experimental variations in the temperature measurements, the samples at 1 mg/mL and control saline (0.9%) were simultaneously exposed to 100 W RF power and imaged using a thermal camera as shown in Figure 3 B. Similar results as in Figure 3 A were observed, with IO-GIC measuring the highest thermal response of ~ 20°C.

Figure 3. (A) Comparative RF heating of IO-GIC NPs as against milli-Q water, 0.9% saline and IO-PAA NPs showing the high heating rate of IO-GIC NPs (B) Comparative thermal images of IO-GIC and IO-PAA NPs at 1 mg/mL and 0.9% saline after RF exposure at 100W for 1 minute (Schematic inserted only to visualize the capacitor position and its coupling to an RF generator) (C) RF heating of IO-GIC NPs as against controls of ‘IO-PAA+GIC’ NP and ‘IO-PAA+EDC’ NP solutions (* p

Gold Ion Nanoprobes for MR Imaging and

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