Ag-doped manganite nanoparticles: New materials for temperature-controlled medical hyperthermia

Ag-doped manganite nanoparticles: New materials for temperature-controlled medical hyperthermia O. V. Melnikov 1, O. Yu. Gorbenko 1 *, M. N. arkelova 1, A. R. Kaul 1, V. A. Atsarkin 2, V. V. Demidov 2, C. Soto 3, E. J. Roy 3, B. M. Odintsov 2 4 1 Chemistry Department, Moscow State University, 119991 Moscow, Russia 2 Institute of Radio Engineering and Electronics RAS, 125009 Moscow, Russia 3 Department of Pathology, College of Medicine, University of Illinois at Urbana-Champaign, 61801 Urbana, Illinois 4 Biomedical Imaging Center of the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 61801 Urbana, Illinois email: O. Yu. Gorbenko ([email protected]) *

Correspondence to O. Yu. Gorbenko, Chemistry Department, Moscow State University, 119991 Moscow, Russia Funded by: Human Frontier Society Program; Grant Number: RGP 047/2007 US National Cancer Institute; Grant Number: R21 CA110010-01 Russian Foundation for Basic Research; Grant Number: 05-02-16371, 07-03-01019 Keywords controllable hyperthermia • nanoparticles • adjustable Curie temperature Abstract The purpose of this study was to introduce newly synthesized nanomaterials as an alternative to superparamagnetic ironoxide based particles (SPIO) and thus to launch a new platform for highly controllable hyperthermia cancer therapy and imaging. The new material that forms the basis for this article is lanthanum manganite particles with silver ions inserted into the perovskite lattice: La1-xAgxMnO3+ . Adjusting the silver doping level, it is possible to control the Curie temperature (Tc) in the hyperthermia range of interest (41-44°C). A new class of nanoparticles based on silver-doped manganites La1-xAgxMnO3+ is suggested. New nanoparticles are stable, and their properties were not affected by the typical ambient conditions in the living tissue. It is possible to monitor the particle uptake and retention by MRI. When these particles are placed into an alternating magnetic field, their temperature increases to the definite value near Tc and then remains constant if the magnetic field is maintained. During the hyperthermia procedure, the temperature can be restricted, thereby preventing the necrosis of normal tissue. A new class of nanoparticles based on silver-doped manganites La1-xAgxMnO3+ was suggested. Ag-doped perovskite manganites particles clearly demonstrated the effect of adjustable Curie temperature necessary for highly controllable cellular hyperthermia. The magnetic relaxation properties of the particles are comparable with that of SPIO, and so we were able to monitor the particle movement and retention by MRI. Thus, the new material combines the MRI contrast enhancement capability with targeted hyperthermia treatment. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009 Received: 30 October 2007; Revised: 14 April 2008; Accepted: 25 April 2008 Digital Object Identifier (DOI) 10.1002/jbm.a.32177 About DOI

Article Text INTRODUCTION

Malignant cells may be more sensitive to heat than normal cells.[1] Thus, raising the temperature within the tumor may provide some selectivity in the destruction of cancer cells. Over the past decade, a method of localized hyperthermia has been developed, which takes advantage of the noninvasive nature of electromagnetic heating. The latter uses ferromagnetic particles that are introduced into the tumor to localize the heating.[2] Although much early work was performed using large implants, which were 1 cm long and 1 mm wide,[3][4] more recently, the emphasis has shifted to using small ferromagnetic and superparamagnetic particles.[5-8] There are different mechanisms providing localized heating within the particles.[9] The amount of heat generated depends upon the frequency and amplitude of the electromagnetic field, the physical properties of the ferromagnetic particles, and their distribution within the tissue. Hilger showed that the temperature rise in the tissue is proportional to the third power of the amplitude of the magnetic field.[5][6] Hergt et al.[9] investigated different particle sizes and shapes and determined that the magnetic hysteresis losses increase with decreasing particle size. Modern clinical hyperthermia trials are based mostly on superparamagnetic iron oxide (SPIO) nanoparticles. The temperature attained within the tumor needs to be within a fairly narrow window (41-43°C) as the higher temperatures may lead to necrosis in the normal tissue, and lower temperature yields recurrent tumor growth. The limitation of iron oxide particles is that the heat generated depends on the density of the iron oxide particles and the power of the applied magnetic fields. In practice, the heat generated varies over a wide range, even within a particular tumor. Thus, there is a compelling need for a new physical concept that may offer reliable temperature control in the targeted tumor volume. Manganite perovskites of the general formula R1-xAxMnO3+ (where R is rare earth cation and A is doping cation) were intensively studied, in the last decade, as materials possessing colossal magnetoresistance. Many attempts were made to find out the doping method that would provide the highest sensitivity of the electrical resistivity to the magnetic field near the room temperature for the application of manganites as magnetic field sensors. Unfortunately, this sensitivity drops rapidly with the increase of the Curie temperature (Tc) for the manganites doped with the bivalent cations such as Ca2+, Sr2+, or Pb2+.[10-13] The electrical and magnetic properties of the manganite ceramics doped with Ca2+ or Sr2+ were significantly improved by the addition of silver.[14-16] The result was related to the enhanced crystallization of the perovskites as well as easier oxygen transport, thus providing a more homogeneous composition. Attempts to insert Ag+ into the La1-xMnO3+ lattice were also described.[17-19] In particular, Raveau and coworkers[20] have reported very high magnetoresistance of silver-doped ceramics at room temperature. On the other hand, the possibility of silver doping in the crystalline structure of perovskite manganites was set in doubt in ref.[21] Only indirect evidences of Ag-doping, such as the absence of metallic silver among the reaction products, have been reported in refs.[20],[22]. Recently, Kaul and coworkers[23] have provided direct evidences for silver doping into the La1xMnO3+ lattice and demonstrated that Ag-doped perovskite manganites possess a broad homogeneity area between La1-xMnO3+ and La1-xAgxMnO3+ compositions (0.3 x 0). It is noteworthy that the formulas are conventional simplification. No oxygen interstitials are

available in the perovskite structure. Apparent excess of oxygen means virtual cation vacancies in the structure. Detailed discussions of the related issues were published recently.[24][25] It was shown that it is possible to control Tc in the hyperthermia range of interest by adjusting the silver content (x) in La1-xAgxMnO3+ . The neutron diffraction refinement of La0.8 Ag0.2MnO3.00 clearly proved the doping of silver. The vacancies in the La-sublattice of the perovskite structure are filled by Ag+ ions under soft synthesis conditions. Note that the Curie temperature exceeds room temperature with the increase of silver content. New nanoparticles are stable, and their properties are not affected by typical ambient conditions in the living tissue. In particular, the oxygen in the surrounding media has no effect on the basic characteristics of La1-xAgxMnO3+ (LaAgMnO) particles. The results show that it is possible to monitor the particle uptake and retention by MRI. The goal of this article is to introduce newly synthesized nanomaterials as an alternative to SPIO-based particles and, thus, launch a new platform for controllable hyperthermia cancer therapy and imaging. EXPERIMENTAL PROCEDURES

The synthesis of uniform nanocrystalline inorganic particles requires a specific chemical protocol. Particularly, the synthesis of oxides with perovskite crystal structure should be conducted at high temperature ( 700°C). This creates difficulties in terms of shape and individual properties of particles. We tried different soft chemical methods for the synthesis of La1-xAgxMnO3 particles. In particular, paper synthesis, synthesis in NH4NO3 melt, and synthesis in nanoreactors and spray pyrolysis were used. All methods use La(NO3)3, Mn(NO3)2 and AgNO3 as precursors. Paper synthesis First, ash-free paper was soaked with the water solution of La, Ag, and Mn nitrates, mixed in the proper ratio, and dried in air at 120°C. Then, paper was burned, and the ash was annealed in the air at 600°C for 30 min; the resulting powder was pressed in pellets. In addition, the silver-rich powder (La/Ag/Mn atomic ratio 0.7/0.4/1) was prepared by the same method. This powder was used as a covering to prevent the loss of silver from the pellets. The silver-containing pellets were sintered in air at 700°C or 800°C under the layer of the covering powder in alundum crucibles for 20 h.[26] Synthesis in NH4NO3 melt La, Ag, and Mn nitrates were mixed, with NH4NO3 as the melting agent. Then, the mixture was heated up to the melting point. NH4NO3 was decomposed, and the solid residue was obtained. The powder was annealed at 700°C in oxygen atmosphere for 5 h. Synthesis in nanoreactors Two microemulsions were prepared as described in ref.[27] from La, Ag, and Mn nitrates solution. Then, the hydroxides were precipitated inside the micelles. TEOS (98%) was added to produce the SiO2 core on the hydroxide particles. After the hydrolysis of TEOS, the resulting particles were centrifuged and dried for 3 h at 160°C. The obtained powder was annealed in oxygen atmosphere at 700°C for 5 h.

Spray pyrolysis A spray of La, Ag, and Mn nitrates water solution was generated by an ultrasonic applicator. The spray mixed with oxygen was admitted through a tubular oven heated up to 1000°C. The effluent powder was captured on a glass filter of pore size less than 100 nm and annealed in oxygen atmosphere at 700°C for 5 h. Materials tests The materials characterization was done by X-ray diffraction (using Rigaku D/MAX-2500 diffractometer, radiation Cu K ) and SEM [using high resolution scanning electron microscope LEO SUPRA 50VP (Carl Zeiss, Germany) equipped with a field emission cathode]. For the SEM study, thin copper plates were covered by water suspension of the manganite nanoparticles and dried in air. An accelerating voltage of 5-10 kV and a magnification 20,000-100,000× were applied. Chemical composition control was accomplished by EDX (using PGT system) and ICP (using atomic emission spectrometer Perkin-Elmer Optima 5300). The Curie temperature was determined from the temperature dependence of r.f. magnetic susceptibility ' at the frequency of 110 kHz. The Tc value was calculated as the point of the maximum slope of '(T) curve. An advanced alternating current (AC) magnetic field applicator was constructed in the Biomedical Imaging Center at the University of Illinois for small animal experiments. In contrast to the applicators described in the literature,[5][6] a water-cooled resonance circuit was used to increase the AC field amplitude H1. We used a solenoid made of 10 turns of copper tube (diameter, 6 cm; Q 145 at f = 800 kHz) as a coil. The inductive resonance coil was matched to the output impedance of the power amplifier. The magnetic field amplitude was measured with a pickup coil by means of an oscillograph. The range of the AC magnetic field frequency f varied from 250 kHz to 1 MHz and field amplitude H1 could reach 12 kA/m. An ENI 3200L Broadband RF Power Amplifier (Rochester, New York) was used with a nominal output power of 300 W. The inhomogeneity of the field amplitude across the animal region of interest inside the coil was negligible. To measure temperature changes during exposure to an alternating magnetic field, we used a thermocouple of copper and constantan wires (0.08 and 0.1 mm in diameter, respectively) connected to a multimeter with automatic registration (Omega 6400-K, Omega Engineering). The direct heating of the thermocouple was found to be negligible. Magnetic resonance images were acquired on a vertical imaging scanner (Oxford Instruments, Abington, UK) equipped with a Unity/Inova console (Varian, Palo Alto, CA), operating at 14.1 T and dedicated to small animal studies. The gradient coils were driven by a set of Varian amplifiers, creating a maximum field gradient of 950 mT/m at 200 V and 100 A with a rise time of 15 s and a deviation from linearity of less than 5%. The mouse was anesthetized and placed in a custom-built animal handling system, which interfaces with a Varian transmitter/receiver quadrature radiofrequency coil of 3.0 cm inner diameter. Mice were imaged 4 h after injection of nanoparticles using a T2-weighted spin-echo pulse sequence, with a slice thickness of 500 m. RESULTS AND DISCUSSION Composition and microstructure

The new material that forms the basis for this article is lanthanum manganite particles with silver ions inserted into the perovskite lattice La1-xAgxMnO3+ . By adjusting the silver doping level (x), it is possible to control their Curie temperature in the hyperthermia range of interest. At the Curie temperature, the magnetic particles lose the ferromagnetic ordering and stop heating. This magnetic phase transition is reversible. After the particles cool off, they become ferromagnetic again and begin to heat up. Thus, when these particles are placed into an alternating magnetic field, their temperature increases to the definite value near Tc. Then, it remains constant, if the magnetic field is maintained. Samples of all La1-xAgxMnO3+ compositions had a rhombohedral perovskite crystal structure. Only paper synthesis and spray pyrolysis produced single-phase perovskite with nearly the same lattice parameters. Also only such samples revealed cation stoichiometry reproducing the cation ratio in the starting solution according to EDX and ICP (synthesis in NH4NO3 melt and nanoreactors resulted in the partial loss of silver and the appearance of the Mn3O4 admixture; Fig. 1). On the other hand, only spray pyrolysis resulted in spherical nanometric particles (Fig. 2). The mean particle diameter was adjusted in the range of 0.1-1 m simply by the variation of the nitrate solution concentration. Figure 1. X-ray phase analysis of La1-xAgxMnO3+ samples prepared by different methods: (1) synthesis in nanoreactors; (2) paper synthesis; (3) synthesis in NH4NO3 melt; (4) spray pyrolysis. [Normal View 24K | Magnified View 59K] Figure 2. Scanning electron microscopy picture presenting microstructure of La1-xAgxMnO3+ , prepared by different methods. All synthesis has been done at 700°C and 1 bar O2 pressure. (c) and (d) demonstrate round particles (numbers in the brackets show the precursor solution molarity). [Normal View 68K | Magnified View 202K]

Magnetic testing The ferromagnetic transition temperature of new nanoparticles was determined from the temperature dependences of r.f. magnetic susceptibility . The real part ( ') was measured in the frequency range of 400-800 kHz. The susceptibility value was determined as an RF generator frequency shift while the generator coil was loaded with nanoparticles. Figure 3 shows the typical temperature dependence of the magnetic susceptibility ( ) of the La0.8 Ag0.15MnO3+ sample made by paper synthesis at 700°C. Characteristic transition temperatures determined from magnetic susceptibility curves are listed in Table I. One can see that the magnetic transition temperature increases with the silver content (y) in the La0.8 AgyMnO3+ composition series. The synthesis temperature increase provides narrowing of the magnetic transition width judged by the TA - TB difference. The results clearly demonstrate that the Curie temperature of La1-xAgyMnO3+ can be adjusted to the hyperthermia interval of 41-44°C by the synthesis conditions. Figure 3. Temperature dependence of the r.f. magnetic susceptibility ( ') of the La0.8Ag0.15MnO3+ sample prepared by paper synthesis at 700°C. Arrows indicate different estimations of the Curie temperatures (TA and TB). [Normal View 19K | Magnified View 43K]

Table I. Transition Temperatures Determined from Magnetic Susceptibility Data

Composition La0.8MnO3 La0.8Ag0.05MnO3 La0.8Ag0.1 MnO3 La0.8Ag0.15MnO3 La0.8Ag0.2 MnO3 La0.8Ag0.15MnO3 La0.8Ag0.2 MnO3

Tsynth (°C)

TA (°C)

TB (°C)

Tstab (°C)

700 700 700 700 700 800 800

20 37 43 45 47 48 48

19 32 40 41 42 47 47

32 42 44 43 47 47

TA and TB indicate different estimations of the Curie temperatures.

Alternating current magnetic field heating Evaluation of the adjustable Tc material for local hyperthermia applications required a comparative analysis of the heating process in the newly synthesized LaAgMnO particles and SPIO nanoparticles both exposed to alternating magnetic fields. Commercially available iron oxide ferrofluid, with a high content of magnetite (Fe3O4) beads (Polysciences Inc., Warrington, PA, Cat 19634) having a particle size of 0.01 m, and 5% solids was used as a reference. An advanced AC magnetic field applicator described in the Experimental section has been used for this testing. Figure 4 shows an essential difference in the AC heating behavior of regular SPIO and LaAgMnO nanoparticles. The behavior of SPIO particles in our experiments is in agreement with the literature data. On the other hand, the effect of lower Tc temperatures of LaAgMnO particles was clearly observed in water suspension. The effect of AC heating temperature stabilization is evident in the silver-doped manganite samples. Note that the AC heating of LaAgMnO particles in Figure 4 is correlated with their r.f. magnetic susceptibility ( ') temperature dependence in Figure 3. Typically, the stabilization temperature Tstab correlated with the characteristic temperatures of the magnetic susceptibility curves (TA and TB): TA > Tstab TB (see Table I). Figure 4. Comparative heating of iron oxide and LaAgMnO nanoparticles in aqueous suspensions when exposed to alternating magnetic fields (H1 = 10 kA/m; f = 800 kHz). Effect of Curie temperature adjustment is clearly seen in the LaAgMnO samples with different Ag content. [Normal View 16K | Magnified View 35K]

There are different mechanisms by which localized heating occurs within magnetic particles, including loss processes by the reorientation of the internal magnetization or frictional losses if the magnetic particle rotates in low-viscous environments. Heat is then transferred to the tissue via thermal conduction.[28] The amount of heat generated depends upon the frequency and amplitude of the alternating magnetic field and upon the size, shape, and microcrystalline structure of the particles, as well as their distribution within tissue. The specific adsorption rate (SAR) is used to describe the magnetic energy amount converted into the heat per time and sample mass.[29] SAR can be estimated by the formula:

where H1 and f are the magnetic field amplitude and frequency, correspondingly; k is a material parameter specific for a given H1f combination. A detailed mechanism of magnetic field energy absorption in the LaAgMnO particle is difficult to predict correctly in terms of Brownian or Neel rotation models, which are applicable for magnetic particles only well below Tc. Biocompatibility testing Biocompatibility testing of new nanomaterials was performed in vitro. It was found that the degradation of La1-xAgyMnO3+ compounds in aqueous suspensions is practically negligible and starts with doped Ag-cations escaping (washing out) into bulk water. It is well known that the presence of Ag-cations in the surrounding is not harmful for living cells and tissues, thus suggesting good biocompatibility of these new materials. We have found that the magnetic properties of new nanoparticles are not affected by ambient conditions in living tissue. Nanoparticles directly implanted into mouse brains did not affect mouse activity. Cells loaded with LaAgMnO nanoparticles were also tested. BV2 cells (a brain microglia cell line) were incubated in vitro with LaAgMnO nanoparticles. Then the cell suspension was passed through a Miltenyi cell sorter with a permanent magnet with magnetic field strength of 0.1 T. Photomicrographs in Figure 5 show (a) empty cells not retained by magnet and (b) microglia loaded with magnetic nanoparticles and retained by the magnet and eluted after removal from the magnetic field. A concentration of 1 g/mL was sufficient to separate the particles by a magnetic sorter.

Figure 5. Brain microglia BV2 cells incubated in vitro with LaAgMnO particles: (a) not retained by the magnet; (b) retained by the magnet. [Normal View 25K | Magnified View 74K]

MRI testing We have done an assessment of whether LaAgMnO nanoparticles would be detectable by MRI in vivo. The mouse was anesthetized with Avertin and placed in a custom-built animal handling system, which interfaced with a transmitter/receiver radiofrequency coil. Twenty 500- m thick slices were acquired with a data matrix of 256 × 256 points, resulting in an in-plane resolution of 100 × 100 m. A spin-echo sequence was used with TR = 0.7 s and TE = 20 ms, with two signal averages giving a total data acquisition time of 6 min. Although gradient-echo sequences are more sensitive to the presence of paramagnetic impurities, the spin-echo images provided improved the anatomical localization of the LaAgMnO particles. Two types of MRI experiments have been done. First, 0.3 L of LaAgMnO nanoparticles in saline has been directly implanted into the mouse brain. In vivo imaging in Figure 6 clearly demonstrates reduction in signal intensity in the area of LaAgMnl particles (0.3 L in saline) directly injected into the mouse brain compared with saline injected in the contralateral side of the brain. The effect of such an enhancing rim was previously reported in ref.[30] Note that nanoparticles have been implanted for many hours before the imaging session and did not affect mouse activity.

Figure 6. In vivo imaging of manganite LaAgMnO nanoparticles (0.3 L), directly implanted into mouse brain, clearly demonstrates reduction in signal intensity (arrow) when compared with that injected with saline in the contralateral side of the brain. [Normal View 27K | Magnified View 82K]

In another experiment, a tumor-bearing mouse was injected with LaAgMnO particles intravenously. Histological section and MRI image of the mouse brain in Figure 7 show that the sensitivity of the MRI imaging is great enough to allow the imaging of a small number of LaAgMnO nanoparticles infiltrating a tumor in the brain. Figure 7. Histological section and MRI image of mouse brain. Mouse was injected with LaAgMnO particles intravenously. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] [Normal View 24K | Magnified View 79K]

The results demonstrate that new nanoparticles are detectable by MRI in vivo, and the MRI can be effectively employed to determine the optimal time point when accumulation of intravenously injected LaAgMnO particles-loaded cells is maximal in the tumor. CONCLUSIONS

A new class of nanoparticles based on silver-doped manganites La1-xAgxMnO3+ was suggested. The important feature of newly synthesized manganites is the possibility to control their Curie temperature in the range of tumor cells hyperthermia interest (41-44°C) by adjusting the silver doping level. Ag-doped perovskite manganites particles clearly demonstrated the effect of adjustable Curie temperature necessary for highly controllable cellular hyperthermia. When these particles are placed into an alternating magnetic field, their temperature increases to the definite value near Tc and then remains constant if the magnetic field is maintained. The magnetic relaxation properties of the particles are comparable with that of SPIO, and so we were able to monitor the particle movement and retention by MRI. Thus, the new material combines the MRI contrast enhancement capability with targeted hyperthermia treatment. Acknowledgements

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