Tumor Biol. DOI 10.1007/s13277-013-1447-y
Dextran-functionalized magnetic fluid mediating magnetohyperthermia for treatment of Ehrlich-solid-tumor-bearing mice: toxicological and histopathological evaluations Ana Luisa Miranda-Vilela & Kelly Reis Yamamoto & Kely Lopes Caiado Miranda & Breno Noronha Matos & Marcos Célio de Almeida & João Paulo Figueiró Longo & José de Souza Filho & Juliana Menezes Soares Fernandes & Patrícia Pommé Confessori Sartoratto & Zulmira Guerrero Marques Lacava
Received: 16 July 2013 / Accepted: 19 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract Dextran-functionalized maghemite fluid (DexMF) has been tested to treat Ehrlich-solid-tumor-bearing mice, evidencing its potential use in mediating magnetohyperthermia in breast cancer treatment. However, although magnetic nanoparticles tend to accumulate in tumor tissues, part of the nanomaterial can reach the blood stream, and then the organism. The aim of this study was to investigate the acute systemic effects of the intratumoral injection of DexMF mediating magnetohyperthermia in the treatment of an advanced clinical Ehrlich-solid-tumor, assessed through histopathological analyses of liver, kidneys, heart and spleen, comet assay, micronucleus test, hemogram, and serum levels of bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, alkaline phosphatase, creatinine, and urea. The tumor’s histopathology and morphometry were used to assess its aggressiveness and regression. DexMF mediating hyperthermia was effective in containing tumor aggressiveness and in inducing tumor regression, besides showing no toxic effects. Its
A. L. Miranda-Vilela (*) : K. R. Yamamoto : B. N. Matos : M. C. de Almeida : J. P. F. Longo : J. de Souza Filho : J. M. S. Fernandes : Z. G. M. Lacava (*) Institute of Biological Sciences, Department of Genetics and Morphology, University of Brasilia, Brasília, Brazil e-mail: [email protected]
e-mail: [email protected]
A. L. Miranda-Vilela Faculty of Medicine, Faciplac, Campus Gama/DF, Brasília, Brazil K. L. C. Miranda : P. P. C. Sartoratto Chemistry Institute, Federal University of Goiás, Goiânia, Brazil
physical characteristics also suggest that it is safe to use in other biomedical applications. Keywords Dextran-coated maghemite nanoparticles . Nanobiotechnology . Magnetic nanoparticles . Magnetohyperthermia
Introduction Breast cancer is the second most frequent worldwide and the most common type of cancer among women, being responsible for about 1.38 million new cases each year, and contributing to about 458,000 deaths, according to the World Health Organization . It is a complex and heterogeneous disease, comprising multiple tumor entities associated with distinctive histological patterns, different biological features, clinical behaviors, and response to therapy [2, 3]. Despite the emergence and evolution of new therapeutic approaches such as conservative surgery, chemotherapy, radiotherapy, hormonal therapy, and immunotherapy in the last half century [4, 5], current treatments for breast cancer remain limited , and breast cancer still continues to be the commonest cause of cancer death among women worldwide . Although nowadays chemotherapy is the most commonly used treatment in the therapeutic approach , chemotherapeutic agents have characteristics that limit their clinical use, including water insolubility, nonspecific biodistribution and targeting, and systemic toxicity . The last of these is responsible for their adverse effects and tend to worsen quality of life, which can compromise the treatment [4, 9]. Also, drug
resistance can develop shortly after initial treatment, limiting the efficacy of therapy . These facts explain the intense scientific research for alternative methods to minimize the toxic and systemic effects of cancer treatments or provide greater specificity to the neoplastic tissue . Among these methods, nanotechnology has been indicated as a new approach to cancer treatment over the past decade [10–12], working towards the development of nanovectors, such as nanoparticles (NPs), that target cancerous cells with drugs and imaging agents, and nanosensors that can detect biological signs of cancer . Thenceforward, increased investigations and developments have been observed in this field, where the use of nanosized magnetic particles has aroused growing interest, given the attractive possibilities these present, such as in improving diagnostic imaging, and as a site-specific drug delivery system [10, 11]. A further application of magnetic nanoparticles (MNPs), magnetohyperthermia (MHT), lies in the production of controlled heating effects  and has emerged as a promising and innovative way to produce important advances in cancer therapy [9–11]. The potential of this therapy as a treatment for cancer was first predicted following observations that several types of cancer cells were more sensitive to temperatures above 41 °C than their normal counterparts . However, while for some types of cancer, such as gliomas, this therapy has already been tested in in vivo and ex vivo experiments in humans , for breast cancer, there are few reports about the antitumor effects of MHT, and these only in animal models [9, 15], so that the role of nanoparticles as drug carriers is the closest nanooncology has come to the patient’s bedside . In the presented context, a dextran-functionalized maghemite fluid (DexMF) has been tested to treat Ehrlichsolid-tumor-bearing mice intratumorally, and results evidenced its potential use in mediating MHT in breast cancer treatment . The enhanced accumulation and retention of DexMF in tumors occurs due to their leaky vasculature and poor lymphatic drainage . However, after tumor treatment, a series of inflammatory mediators , as well as the remaining magnetic nanoparticles, can reach the vascular system due to absorption by the lymphatic capillaries, and consequently, the organs . After the possible dissemination of the nanomaterials by the organism, a series of complex and factorials bio-physical–chemical interactions can happen , making it essential to investigate its potential biological undesirable side effects through histology and biochemical evaluations, especially with a view to DexMF use in future clinical trials. The aim of this study was to investigate the acute systemic biological effects of the intratumoral injection of DexMF used to mediate magnetohyperthermia in the treatment of an advanced clinical Ehrlich-solid-tumor in female Swiss albino mice.
Materials and methods Chemicals Ketamine chloridrate, sold as Dopalen 100 mg/mL, was obtained from Ceva Animal Health Ltda (São Paulo, Brazil); xylazine chloridrate (Coopazine® 20 mg/mL) came from Coopers (São Paulo, Brazil); FeCl3.6H2O and dextran of high molecular weight (250 kDa) from Acros Organics (Geel, Belgium); and FeCl2.4H2O from Fluka Analytical (SigmaAldrich, São Paulo, Brazil).
Magnetic fluid The magnetic fluid (DexMF) used to carry out the experiments was based on dextran-coated maghemite nanoparticles dispersed in water. Coated nanoparticles with core size of 6.6± 2.7 nm average diameter  were synthesized by the method of co-precipitation of Fe (II) and Fe (III) in an alkaline medium, with the reaction occurring in the presence of polysaccharide dextran of 250 kDa molecular weight, as described below.
Synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) The first step was the synthesis of magnetite nanoparticles (Fe3O4), using Massart’s co-precipitation method , with the reaction occurring in the presence of polysaccharide dextran of 250 kDa molecular weight. In the synthesis, a solution containing iron salts (5.4 g FeCl 3 .6H 2 O and 2,185 g FeCl2.4H2O dissolved in 70 mL of water) was added to a solution of dextran (1.240 g dextran in 150 mL deionized water), and the mixture subjected to mechanical stirring for 20 min. Then 50 mL of ammonia solution (28 %) was added to the mixture containing dissolved iron salts and polysaccharide, with the immediate precipitation of a black and magnetic solid being observed, characteristic of iron oxide Fe3O4. The system was maintained under mechanical stirring for more than 20 h to allow efficient adsorption of the polysaccharide to the surface of magnetic nanoparticles. The obtained solid (Fe3O4 nanoparticles coated with dextran) was isolated by centrifugation (3,500 rpm) and then washed successive times (in order to eliminate impurities and free dextran) with deionized water, until the pH of the supernatant remained at about 7. The carbon content in the sample (powder) was determined in an organic elemental analyzer CHNSO Flash 2000 (Thermo Scientific). The analysis of the iron content was performed by the colorimetric orthophenanthroline method, and the absorbance measured at wavelength of 515 nm in a U-1100 UV/Visible spectrophotometer (Hitachi High-Technologies Corporation).
Preparation of the magnetic fluid based on dextran-coated maghemite nanoparticles (DexMF) The DexMF preparation consisted of adding to the moistened solid (Fe3O4/dextran) 80 mL of deionized water, with the mixture being submitted to ultrasonic irradiation for 20 min. The colloidal dispersion obtained was centrifuged for 20 min at 4,500 rpm, and the precipitate discarded, rendering a stabilized fluid with 6.6±2.7 nm average-diameter maghemite nanoparticles coated with dextran in a concentration of 1.5× 10−2 g Fe/mL, as previously reported . The obtained supernatant (DexMF) was sterilized by filtration through a 0.220-μm filter before use. Zetasizer equipment (Zetasizer Nano-ZS90 Malvern Instruments Limited, Malvern, UK) was used to determine the hydrodynamic average diameter of maghemite nanoparticles by dynamic light scattering (DLS), its polydispersion index (PDI), and its surface charge by measuring the zeta potential. For these, approximately 1 mL of the diluted solution at the ratio 1:20 in distilled water was used. Alternatively, different pH aqueous solutions were used to evaluate the influence of pH on the hydrodynamic diameter, polydispersion index and zeta potential. Analyses were performed in triplicate at 25 °C, with a fixed detection angle of 173°. To evaluate the influence of serum proteins on the hydrodynamic diameter, the nanoparticles were diluted (1:20) in different concentrations (0; 0.625 %; 1.25 %; 2.5 %; 5 %) of aqueous bovine serum albumin (BSA) solutions for 24 h, in different temperature conditions (25 °C and 37 °C). The stability of the suspension was analyzed over time for more than a year after the experimental period, where the nanoparticles were again evaluated in the Zetasizer equipment, according to the previously described methods. Ehrlich tumor The Ehrlich ascitic tumor, derived from a spontaneous murine mammary adenocarcinoma, was maintained in ascitic form by passages in Swiss mice, by weekly intraperitoneal transplantation as previously described [9, 21]. Animals and experimental design Thirty female Swiss albino mice, 11–12 weeks old, weighing 30.31±2.26 g, were obtained from the Multidisciplinary Center for Biological Investigation in Laboratory Animal Science (CEMIB) of the State University of Campinas (Unicamp, SP/ Brazil). They were randomly housed in plastic cages (6/cage) under standard conditions at 22±2 °C in a 12-h light/dark cycle and fed with standard diet and water ad libitum . After acclimatizing the animals for 2 weeks, they were anesthetized by intraperitoneal administration of ketamine (80 mg/kg) and xylazine (10 mg/kg). A volume of 40 μL
corresponding to 5.5×106 viable cells was directly subcutaneously injected in the upper region of the head according to a previously described procedure , for the solid form induction. Forty-eight hours after tumor cell implantation, all mice had clinical tumor implanted and were treated as follows: (a) tumor inoculation and no treatment (tumor group), (b) intratumoral injection of DexMF (4 μL) once a day for three consecutive days (DexMF group), (c) exposure for 10 min to an alternating current (AC) magnetic field (40 Oe amplitude AC magnetic field oscillating at 1 MHz) once a day for three consecutive days (MF group), and (d) intratumoral injection of DexMF followed by exposure to AC magnetic field under conditions specified in b and c to produce magnetohyperthermia (MHT group) . The magnetic field exposure time and treatment used were previously tested [9, 15] and chosen to be sufficient to induce magnetic hyperthermia with practically no hematological adverse effects. Negative control (NC group) received filtered water and no tumor was implanted. All procedures were reviewed and approved by the institutional Ethics Committee for Animal Research (Institute of Biological Science, University of Brasília), number 107748/ 2009. Due to the requirement of the institutional Ethics Committee for Animal Research to reduce the number of animals used, the tumor group used here also belonged to the previously published study (tumor group, 1 week) , both studies being performed at the same time. Procedures and measurements Body weights were measured at the beginning and end of the treatment, and the animals were monitored for signs of toxicity such as weight loss, diarrhea, skin ulcers, and deaths. Forty-eight hours after the last treatment, animals were anesthetized with the mixture of xylazine and ketamine described above. Blood samples (1 mL) collected by cardiac puncture were used to carry out hemogram, biochemical dosages of total bilirubin, direct bilirubin, indirect bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), alkaline phosphatase, creatinine and urea, as well as the comet assay. After this, euthanasia of the animals was carried out by cervical dislocation, according to the American Veterinary Medical Association (AVMA) guidelines for euthanasia ; the tumor, liver, kidneys, heart, and spleen were surgically removed for histological evaluations, and the bone marrow was extracted to perform the micronucleous (MN) test. Hemogram was processed in a multiple automated hematology analyzer for veterinary use, Sysmex pocH-100iV Diff (Curitiba/Paraná, Brazil) calibrated for mice in microtubes containing EDTA as anticoagulant. Total bilirubin, bilirubin fractions, and creatinine were measured by colorimetric assays; GGT by a colorimetric kinetic method; urea by an enzymatic colorimetric method; and AST, ALT, and alkaline
phosphatase by optimized kinetic methods; all of them running on the automated chemistry analyzer ADVIA 2400 (Siemens), using the appropriate Advia chemistry reagents, protocols and controls. Tumors were weighed; their width, length, and thickness were measured using a digital pachymeter and their volume was calculated . Afterwards, tumors, liver, kidneys, heart, and spleen were fixed with 10 % formalin for 24 h, transferred to 70 % ethanol, included in paraffin using an automatic tissue processor (OMA® DM-40, São Paulo, Brazil), cut to 5 μm of thickness in a Leica RM2235 manual microtome (Leica Microsystems, Nussloch, Germany) and stained with hematoxilin–eosin (HE) for histological analyses (light microscopy). A total of five histological sections with 100 μm distance between sections were analyzed per tumor sample. All histological sections were photographed with MC 80 DX camera coupled to a Zeiss Axiophot light microscope and tumor/ necrotic areas were quantified using Image ProPlus 5.1 software. The comet assay (alkali method), done in situ, was immediately processed according to the standard method  with a few modifications as previously described . The micronucleus (MN) test followed previous protocol  and was used to study DexMF genotoxicity (MN evaluation) and cytotoxicity (frequency of polychromatic erythrocytes—%PCE), respectively. For both tests, comet and MN, a positive control (PC group) group was included, intraperitoneally treated with cyclophosphamide at 25 mg/kg 24 h before euthanasia.
hydrodynamic diameter and polydispersion index were, respectively, 204.3 nm±0.85 and 0.263±0.008, confirming the stability of the formulation in the nanometric size scale, with a reduced aggregation process that could compromise the biodistribution of the active material, and its interaction with the biological target. Moreover, pH variations did not have significant influences on these parameters (Fig. 1a), although, as expected, they significantly impacted the zeta potential of the magnetic nanoparticles (Fig. 1b). In addition, there was an increase in the hydrodynamic diameter of the nanoparticles when these were suspended in different concentrations of
Statistical analyses Statistical analysis was carried out using SPSS (Statistical Package for the Social Sciences) version 17.0. Data were expressed as mean±SEM (standard error of mean) and values of p