Article Journal of Biomedical Nanotechnology

Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 9, 1261–1271, 2013 www.aspbs.com/jbn

Dextran-Functionalized Magnetic Fluid Mediating Magnetohyperthermia Combined with Preventive Antioxidant Pequi-Oil Supplementation: Potential Use Against Cancer Ana Luisa Miranda-Vilela1 ∗ , Raphael Cândido Apolinário Peixoto2 , João Paulo Figueiró Longo1 , Débora de Oliveira Silva e Cintra2 , Flávia Arruda Portilho1 , Kely Lopes Caiado Miranda3 , Patrícia Pommé Confessori Sartoratto3 , Sônia Nair Báo2 , Ricardo Bentes de Azevedo1 , and Zulmira Guerrero Marques Lacava1 1

Department of Genetics and Morphology, Institute of Biological Sciences, University of Brasilia, Brasília, 70.910-900, Brazil Electron Microscopy Laboratory, Department of Cell Biology, Institute of Biological Sciences, University of Brasilia, Brasília, 70.910-900, Brazil 3 Chemistry Institute, Federal University of Goiás, Goiânia, 74001-970, Brazil 2

Delivered by Publishing Technology to: Rice University IP: 188.112.177.181 On: Thu, 04 Feb 2016 20:56:59 This work aimed to test a dextran-functionalized fluid (DexMF)Publishers sample in mediating magnetohyperthermia to Copyright:magnetic American Scientific

treat an advanced clinical Ehrlich-solid-tumor, to verify the effects of oral antioxidant administration of pequi-oil on this treatment and to investigate the potential of these treatments for future use as an adjuvant in cancer therapy. Animals received the treatments: (a) filtered water (control); (b) tumor implantation and no treatment (tumor group); (c) tumor implantation followed by intratumoral injection of DexMF and alternating current magnetic field exposure (MHT group) for three consecutive days; (d) oral pequi-oil supplementation followed by tumor implantation and the same treatment as group MHT (PMHT group). Analyses took place 1 and 2 weeks after tumor implantation. Both treatments were effective in increasing the tumor necrosis process and controlling tumor growth, besides keeping lymphocyte-dependent immunity. Although the MHT treatment was more efficient after the first week in reducing DNA damage to blood peripheral leucocytes, PMHT therapy appeared to be more effective with the advance of the carcinogenesis process after the second week. Our findings evidence the potential use of DexMF mediating magnetohyperthermia in cancer treatment and also suggest that the preventive pequi oil administration could increase the efficiency of this process.

KEYWORDS: Dextran-Coated Maghemite Nanoparticles, Pequi Oil (Caryocar Brasiliense Camb), Nanobiotechnology, Magnetic Nanoparticles, Magnetohyperthermia.

INTRODUCTION Cancer is one of the most challenging health problems worldwide and is among the most important causes of morbidity and mortality.1 2 Due to its many different causes and distinct characteristics, cancer requires different treatments. The classically adopted treatments for cancer have significant importance in increasing patient survival, ∗

Author to whom correspondence should be addressed. Emails: [email protected], [email protected] Received: 6 June 2012 Revised/Accepted: 6 January 2013

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but they may also present numerous acute and chronic adverse effects, compromising treatment.1–3 These facts explain the intense scientific research for alternative methods to minimize the toxic and systemic effects of cancer treatments or provide greater specificity for neoplastic tissue. In this context, nanobiotechnology approaches and in particular the use of magnetic nanoparticles (MNPs) have emerged as promising and innovative ways to produce important advances in cancer therapy, such as magnetohyperthermia (MHT).4–6 Biocompatible MNPs may generate heat when submitted to an external alternating current magnetic field.4 5 The heat generated

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raises the local temperature to 41–42  C and has the effect pequi oil supplementation associated with magnetohyperthermia could reduce the carcinogenic process, controlling of selectively destroying tumor cells, since these are less tumor growth. Because magnetic fluid based on dextranresistant to sudden increases in temperature than the surcoated nanoparticles has been well studied as to its physirounding normal cells.4 Indeed, several interesting reports cal and biological properties,31–35 it seems to be a suitable about the effects of MHT have been published (5, 7–10), 7 8 magnetic fluid to test MHT in tumor control. Thus, the aim including antitumor effects. of this study was to test dextran-functionalized maghemite However, the use of MHT technology raises the question fluid (DexMF) in mediating MHT to treat the inducible of possible adverse effects on the organism of magnetic carcinogenic process in Ehrlich-solid-tumor-bearing mice, field exposure, which may depend on exposure time.5 9 10 to verify the effects of oral supplementation with pequi oil Also, because biomedical applications of MNP are usuon magnetohyperthermia-induced reactions and to investially based on magnetite or maghemite nanoparticles,4 5 gate the potential of these treatments for future use as an accumulation of iron may occur during treatment and, adjuvant in cancer therapy. Considering that breast cancer when present in excess, iron can harm biological sysis the most common cause of cancer death among women tems since in redox-active form it catalyzes the gen11–13 worldwide36 and the available therapies cause acute and eration of highly reactive oxygen species (ROS). chronic adverse effects that compromise the treatment and Thus, combining MHT with nutritional therapy based on patients’ survival, it is necessary and urgent to develop antioxidants could increase cancer treatment efficiency by new proposals that could minimize the toxic and systemic protecting against harmful effects. This is because, while effects of conventional treatments. oxidants may contribute to the carcinogenesis process by causing DNA damage, suppressing lymphocyte-dependent immunity and stimulating cell division, antioxidants have MATERIALS AND METHODS important antitumor effects, such as neutralizing oxidants, Magnetic Fluid decreasing oxidative DNA damage, restoring lymphocyteThe magnetic fluid sample based on dextran-coated mediated defense, and reducing cell divisions.13 maghemite nanoparticles (DexMF) was used to carry out The oil extracted from pequi (Caryocar brasiliense the experiments. DexMF was obtained by chemical coCamb.), a typical fruit of the Brazilian Cerrado, is rich precipitation of Fe(II) and Fe(III) ions that yielded magDelivered Publishing Technology Rice University in various carotenoids, several of them by with pro-vitamin netite to: nanoparticles in the presence of high molecular IP: 188.112.177.181 On: Thu, 04 Feb 2016 20:56:59 A activity,14–16 and all of them presenting very effecweight dextran (250 Copyright: American Scientific Publishers kDa) in an alkaline medium. The tive antioxidant properties under low O2 tension (PO2 .2 magnetite oxidized to maghemite during the purificaAlthough under high PO2 , they are less efficient,2 prestion process of washing the precipitate with deionized ence of vitamin E (-tocopherol) has been also reported water until the supernatant achieved neutral pH. After even in the cooked pulp,17 and this vitamin is an efficient centrifugation, the precipitate was dispersed in deionized antioxidant for cells submitted to high PO2 .2 Carotenoids water by sonication rendering the stabilized fluid with have been reported to reduce the risk of breast cancer, 66 ± 27 nm average-diameter maghemite nanoparticles while vitamin A (retinol) and retinoids appear to inhibit the coated with dextran (Fig. 1) in a concentration of 15 × growth of certain cancer cell lines, and vitamin E has been 10−2 g Fe/mL. The hydrodynamic diameter and polydispersion index were 168 nm and 0.144, respectively. seen to reduce the risk of some forms of cancer, including 2 13 18 The fluid samples were sterilized by filtration through a prostate and colon cancer, pequi oil, which contains 0.220 m filter before use. these three bioactive compounds, could favor the inhibition 19 of tumor growth, if preventively administered. In fact, pequi oil has been reported as having important antioxidant Chemicals activity in vitro20 and in vivo,21–24 as well as presenting Ketamine and xylazine were obtained as chloridrate. anti-inflammatory activities,25 besides controlling the carKetamine sold as Dopalen 100 mg/mL was obtained from cinogenesis process induced in mice inoculated with the Ceva Animal Health Ltda (São Paulo, Brazil), and xylazine Ehrlich murine mammary adenocarcinoma.19 Therefore, it (Coopazine® 20 mg/mL) came from Coopers (São Paulo, Brazil). is reasonable to suppose that oral supplementation with pequi-oil could reduce the carcinogenic process and help to control the potential adverse effects of MHT mediated Plant Material by magnetic fluids (MF) containing iron oxide nanopartiPequi fruit was obtained in natura from the local markets cles (e.g., magnetite or maghemite). of Brasília/DF (Brazil) and surrounding areas. The internal Since antioxidant enzyme activity decays in a wide varimesocarp was peeled or grated to obtain the pulp, which ety of tumors26–30 and oxidants are an important class of was packed in a covered pot and frozen at −86  C. Pequi agents that stimulate cell division, cause damage to DNA, pulp oil was extracted by cold maceration using chloroproteins, and lipid, and reduce lymphocyte-dependent form as the solvent. The extract was subjected to evaporation under reduced pressure and dried at high vacuum for immunity, favoring mutagenesis and carcinogenesis,13 oral 1262

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Figure 1. tion (B).

Antioxidant and Magnetohyperthermia Against Cancer

Transmission electron micrography of dextran-coated magnetic nanoparticles (A) and their length-frequency distribu-

complete solvent removal. The relative composition of the pequi fruit pulp oil is shown in Table I.

for Biological Investigation in Laboratory Animal Science (CEMIB) of the State University of Campinas (Unicamp, SP/Brazil), were housed in plastic cages (6/cage) at room temperature (22 ± 2  C) in a 12 h light/dark cycle with Ehrlich Tumor Delivered Publishing Technology lights on to: at 6Rice a.m.University and free access to food and water. The Ehrlich ascitic tumor, derived from by a spontaneous IP: 188.112.177.181 On: Thu,The 04 Feb 2016were 20:56:59 animals anesthetized by an intraperitoneal murine mammary adenocarcinoma, was maintained in Copyright: American Scientific Publishers administration of ketamine (80 mg/kg) and xylazine ascitic form by passages in Swiss mice, by weekly 6 19 (10 mg/kg) solution in a final dose of 0.1 mL/30 g. intraperitoneal transplantation of 10 tumor cells. The Ehrlich ascitic tumor cell suspension was injected subcuascitic fluid was collected by intraperitoneal puncture 7 19 taneously, as previously reported, for the solid form using a sterile insulin syringe. Ascitic tumor cell counts implantation, but injecting now 55 × 106 viable cells were done in a Neubauer hemocytometer. The cells were 4 instead of previously reported 55 × 10 viable cells, to found to be more than 99% viable by the Trypan blue dye produce a more clinically advanced tumor. Mice (N = exclusion method. 6 per group) received the following treatments 48 hours after tumor inoculation: (a) filtered water and no tumor Animals and Experimental Design implantation (negative control, named “Control group”); Female Swiss albino mice, 11–12 weeks old, weighing (b) tumor inoculation and no treatment (control tumor, 312 ± 22 g, obtained from the Multidisciplinary Center named “Tumor group”); (c) intratumoral injection of DexMF (40 L) containing 755 × 1015 nanoparticle/mL Table I. Relative composition of pequi (Caryocar brasiliense and exposure for 10 minutes to an alternating current (AC) Camb.) pulp oil. magnetic field (40 Oe amplitude AC magnetic field oscillating at 1 MHz),9 once a day for three consecutive days Fatty acids37∗ (%) starting 48 hours after tumor inoculation (MHT group); Saturated Mono-unsaturated Poly-unsaturated (d) oral pequi-oil supplementation (30 mg/animal/day) by Palmitic 4178 Oleic 5428 Linoleic 1.36 gavage for 10 consecutive days followed by the treatStearic 128 Palmitoleic 067 Linolenic 0.51 ment described for group MHT (PMHT group). The used Araquidic 012 magnetic field exposure time and treatment was previCarotenoids14–16 ously tested by Portilho et al. 20117 and chosen here to (mg/100 g of pequi fruit pulp) 17 be sufficient to induce magnetic hyperthermia with practiVitamin E (g/100 g Pro-vitamin A Lycopene Total of pequi fruit pulp) cally no hematological adverse effects. Daily dose of the pequi-oil was calculated using the dose translation for6.26–11.5 1.12–2.08 6.75–28.66 > 110.52 mula developed by Reagan-Shaw et al. (2007),38 obeyNote: ∗ Present study. ing the maximum daily dose of provitamin A carotenoids J. Biomed. Nanotechnol. 9, 1261–1271, 2013

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(25 mg) recommended by the National Agency for Sanitary Surveillance (ANVISA). To verify the effectiveness of the treatments over time, all five groups were duplicated and analyses took place one or two weeks after tumor inoculation. Euthanasia was carried out by cervical dislocation. To avoid adverse effects on the organism as function of the magnetic field exposure time,5 9 10 this one was

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previously tested by Portilho et al. (2011)7 and in the present study, and the best exposure time was chosen (exposure for ten minutes once a day for three consecutive days), which was sufficient to induce magnetic hyperthermia without causing adverse effects. All procedures described were reviewed and approved by the institutional Ethics Committee for Animal Research

Delivered by Publishing Technology to: Rice University IP: 188.112.177.181 On: Thu, 04 Feb 2016 20:56:59 Copyright: American Scientific Publishers

Figure 2. Tumor histology (A), (B), tumor volume (C), (E) and percentage of necrosis area (D), (F) before and after MHT and PMHT treatments in the first and second weeks after tumor inoculation. Notes: Bar graphs were expressed as SEM (standard error of mean). N = Necrosis area; V = viable tumor tissue; MNP = magnetic nanoparticles. (A) Histology of non-treated tumor: small, higher chromatophilic tumor cells of variable shape with intact cell nucleus and cytoplasm limits represent viable tumor (V) surrounding areas of necrosis (N), represented by an amorphous eosinophilic tissue without cytoplasmatic membrane integrity and absence of cell nucleus. (B) Histology of tumor treated with MNP and AC magnetic field to produce magnetohyperthermia. (C) Tumor volume at 1 week. (D) Necrosis area of tumor at 1 week. (E) Tumor volume at 2 weeks. (F) Necrosis area of tumor at 2 weeks. P -value of tumor volume was generated by ANOVA, while p-value of necrosis area was generated by Kruskall-Wallis test. Asterisks indicate significant differences at p < 0001 detected by Dunn’s Multiple Comparison Tests in respect to the non-treated tumor (Tumor group). The symbol = indicates significant differences between 1 and 2 weeks for the same treatment.

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(Institute of Biological Science, University of Brasília), number 107748/2009. Procedures and Measurements Before euthanasia, animals were anesthetized with the mixture of xylazine and ketamine described above. Blood samples (1 mL) collected by cardiac puncture, using an insulin syringe containing EDTA as anticoagulant, were used to carry out comet assay and hemogram. The latter test was processed in a multiple automated hematology analyzer for veterinary use, Sysmex pocH-100iV Diff (Curitiba/Paraná, Brazil) calibrated for mice. Tumors were removed; their width and length measured by a digital pachymeter, and the tumor volume calculated according to Yanase et al. (1998).39 Afterwards, tumors were fixed with 10% formalin for 24 hours, transferred to 70% ethanol, included in paraffin using an automatic tissue processor (OMA® DM40, São Paulo, Brazil), cut to 5 m of thickness in a Leica RM2235 manual microtome (Leica Microsystems, Nussloch, Germany) and stained with hematoxilin-eosin for histological analyses (light microscopy).

Antioxidant and Magnetohyperthermia Against Cancer

RESULTS There were no significant differences in animals’ body weight among all the analyzed groups. Tumor Histology and Measurements Central areas of necrosis were identified in all experimental groups (Figs. 2(A) and (B)). However, treated tumors differed from non-treated by the presence of necrotic tissue in the peripheral areas. In these groups, necrotic areas were also highly infiltrated by defense cells, especially neutrophils, and there were hemorrhagic regions with red blood cells outside the veins (data not shown). The presence of magnetic nanoparticles was identified

Tumor Necrosis Area Measurements A total of five histological sections, 100 m apart, were analyzed per tumor samples. All histological sections were photographed with MC 80 DX camera coupled to a Zeiss Technology to: Rice University Delivered by Publishing Axiophot light microscope and tumor/necrotic areas were IP: 188.112.177.181 On: Thu, 04 Feb 2016 20:56:59 Copyright: American Scientific Publishers quantified using Image ProPlus 5.1 software. Single-Cell-Gel Electrophoresis (Comet Assay) Blood collection was done in situ and immediately processed for the comet assay (alkali method), which was performed as previously described.19 For this test, a positive control group intraperitoneally treated with cyclophosphamide at 25 mg/kg 24 hours before euthanasia (group “CP”) was included. Statistical Analysis Statistical analysis was carried out using SPSS (Statistical Package for the Social Sciences) version 17.0 and GraphPad Prism version 5.00 softwares. Data were expressed as mean ± SEM (standard error of mean) and values of p < 005 were considered statistically significant. The continuous variables were tested for normal distribution with Kolmogorov-Smirnov. For animals’ body weight, the differences among groups were checked by ANOVA, while differences in other variables were investigated by ANOVA or Kruskal-Wallis test (data not normally distributed), followed, respectively, by Tukey’s or Dunn’s Multiple Comparison Tests. Initially, the results of groups with tumors after one week and two weeks were compared separately. Later, the same results obtained one and two weeks after tumor inoculation were compared between themselves. J. Biomed. Nanotechnol. 9, 1261–1271, 2013

Figure 3. DNA damage in peripheral leucocytes of Ehrlich solid tumor-bearing mice after MHT and PMHT treatments in the first (A) and second (B) weeks after tumor inoculation. Negative control (group “Control”) received filtered water and no tumor implantation took place; positive control (group “CP”) received cyclophosphamide at 25 mg/kg intraperitoneally 24 hours before euthanasia; and in the Tumor group, tumor was implanted and no treatment was performed. Notes: Bar graphs were expressed as SEM (standard error of mean). a.u. = arbitrary units. P -values were generated by ANOVA. The superscript letters indicate significant differences detected by Tukey’s test, with a = significant compared to Control; b = significant compared to CP; c = significant compared to Tumor. Asterisks indicate significant (∗ p < 005) differences. The symbol = indicates significant differences between 1 and 2 weeks for the same treatment.

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as brown-gold spots in the histological sections of the DNA damage compared to the tumor group, a signifitumors. In the animals treated with magnetic nanoparticant decrease in DNA damage was only observed for the cles and subsequently exposed to the AC magnetic field to MHT group. After the second week, decreased but noninduce magnetohyperthermia (MHT and PMHT groups), significant DNA damage was only observed in the PMHT the tumors presented tissue necrosis near the area of magtreatment. The MHT group thus presented a significant netic nanoparticles deposition, but viable tumor cells could difference when first and second weeks were compared be observed a few micrometers (M) from the nanoparti(Fig. 3). cle spot accumulation (Fig. 2(B)). One week after tumor inoculation, in both treatments (MHT and PMHT groups) Hematology the remaining cutaneous tissue presented the swollen tissue In the erythrogram (Table II), one week after tumor typical of edema, which contributes to non-significantly implantation only the mean corpuscular volume (MCV) increased tumor volume compared to non-treated animals of MHT group significantly increased compared to non(Fig. 2(C)). Two of the six animals treated with MHT pretreated animals (Tumor group). After the second week, sented absence of viable tumor tissue: there were spread red blood cell (RBC) numbers were significantly lower islands of differentiated cells immersed in extensive necroin tumor-bearing non-treated (Tumor group) and treated sis regions; cell proliferation areas were absent; the tumors (MHT and PMHT groups) mice than in negative conwere dying. Thus, tumor morphometry was conducted only trol (Control group). The same occurred with hemoglobin in the remaining four animals, and even in these animals (HGB) and hematocrit (HCT) in Tumor and PMHT the necrosis area was significantly higher than in nongroups, while MCV and mean corpuscular hemoglobin treated animals (Fig. 2(D)). Two weeks after tumor inocu(MCH) were significantly higher in both treatments (MHT lation, the histological sections showed viable tissue in all and PMHT) than in the negative control and non-treated treated animals. Non-significantly decreased tumor volume animals. Red cell distribution width (RDW) was also sigwas seen for both treatments, MHT and PMHT, in relation nificantly higher in non-treated and treated Ehrlich-solidto the non-treated tumor (Fig. 2(E)), but the necrotic area tumor-bearing mice. in the treated animal continued to be statistically higher In the leukogram (Fig. 4) results, one week after (Fig. 2(F)). tumor implantation, PMHT treatment induced a significant Delivered by Publishing Technology to:inRice increase the University number of lymphocytes and neutrophils + IP: 188.112.177.181 On: Thu,monocytes 04 Feb 2016 20:56:59 Comet Assay compared to negative control and non-treated Copyright: Cyclophosphamide significantly increased DNAAmerican damage Scientific animals.Publishers After the second week, the percentage of these compared to all other groups. Although after the first cells dropped significantly, but their number remained week both treatments (MHT and PMHT groups) reduced higher than in the negative control. Table II. Erythrogram of Ehrlich solid tumor-bearing mice after MHT and PMHT treatments in the first and second weeks after tumor inoculation. Negative control (group “Control”) received filtered water and no tumor implantation took place. In the group “Tumor,” tumor was implanted and no treatment was performed. RBC (×106 /L)

HGB (g/dL)

HCT (%)

MCV (fL)

MCH (pg)

1 2 3 4

885 ± 016 865 ± 040 772 ± 008 783 ± 026 0.055

1314 ± 019 1254 ± 053 1177 ± 013 1180 ± 032 0.020

3258 ± 052 3104 ± 136 2987 ± 020 2920 ± 076 0.215

3684 ± 031 3596 ± 045 3872 ± 025∗b 3748 ± 139 0.015

1486 ± 016 1454 ± 018 1525 ± 015 1515 ± 060 0.036

5 6 7 8

883 ± 013 801 ± 016∗ a 758 ± 021∗∗ a 737 ± 013∗∗ a 0.001

1307 ± 015 1146 ± 020∗∗ a 1198 ± 030 116 ± 012∗ a 0.007

3235 ± 044 2918 ± 060∗ a 3005 ± 065 2892 ± 042∗ a 0.016

3666 ± 026 1481 ± 013 3643 ± 027 1430 ± 010 3954 ± 029∗∗ a b 1578 ± 017∗∗a b 3928 ± 021∗∗ a b 1577 ± 015∗∗ a b 0.000 0.000

Treatment Group

MCHC (g/dL)

RDW (%)

1 week Control Tumor MHT PMHT P-values 2 weeks Control Tumor MHT PMHT P-values

4036 ± 028 1349 ± 025 4041 ± 035 1409 ± 034= 3938 ± 021 1488 ± 017 4040 ± 022 1705 ± 221 0.122 0.027 4042 ± 023 3928 ± 027 39.92 ± 0.32 4012 ± 031 0.528

1346 ± 019 1532 ± 043∗ a 1550 ± 028∗ a 1500 ± 025∗ a 0.013

Notes: Data were expressed as mean ± SEM (standard error of mean). RBC = Red Blood Cells; HGB = Hemoglobin; HCT = Hematocrit; MCV = Mean Corpuscular volume; MCH = Mean Corpuscular hemoglobin; MCHC = Mean corpuscular hemoglobin concentration; RDW = Red cell distribution width (represents an indication of the amount of variation—anisocytosis—in cell size); g/dL = grams per deciliter; fL = fentoliters; pg = picograms. For 1 week, p-values of RBC, HCT and MCHC were generated by ANOVA, while p-values of the other parameters were generated by the Kruskall-Wallis test. For 2 weeks, p-values of RBC, HCT, MCV, MCH, MCHC and RDW were generated by ANOVA, while p-value of HGB was generated by the Kruskall-Wallis test. The superscript letters indicate significant differences detected by Tukey’s or Dunn’s Multiple Comparison Tests, a = significant compared to Control; b = significant compared to Tumor. Asterisks indicate significant (∗ p < 005) and highly significant (∗∗ p < 001) differences. The symbol = indicates significant differences between 1 and 2 weeks for the same treatment.

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Figure 4. Differential leukocyte counts of Ehrlich solid tumor-bearing mice after MHT and PMHT treatments in the first (A) and (C) and second (B) and (D) weeks after tumor inoculation. Negative control (group “Control”) received filtered water and no tumor implantation took place. In the group “Tumor,” tumor was implanted and no treatment was performed. Notes: Bar graphs were expressed as SEM (standard error of mean). A and C express, respectively, percentage and differential leukocyte counts from the leukogram in the first week after tumor inoculation, while B and D show the second week. P -values of Delivered by Publishing Technology to: Rice University lymphocytes and neutrophils + monocytes were generated by ANOVA, while p-value of eosinophils was generated by KruskallIP: 188.112.177.181 On: Thu, 04 Feb 2016 20:56:59 Wallis test. The superscript letters indicate significant differences detected by Tukey’s or Dunn’s Multiple Comparison Tests, with Copyright: American Scientific Publishers a = significant compared to group Control; b = significant compared to group Tumor. Asterisks indicate significant (∗ p < 005) and highly significant (∗∗ p < 001) differences. The symbol = indicates significant differences between 1 and 2 weeks for the same treatment.

No significant differences were observed in the plateletgram (data not shown).

DISCUSSION For in vivo biomedical applications, magnetic nanoparticles must be made of a non-toxic and non-immunogenic material, with small enough particles to pass through the capillary systems of organs and tissues avoiding vessel embolism.4 These considerations suggest that nanoparticles of about 5–10 nm in diameter should be ideal for most forms of therapy but that there will also be problems of formulating particle concentrations and suspending media to obtain best distributions.6 Also, they must have high magnetization so that they can be immobilized in the targeted pathologic tissue by a magnetic field.4 In this context, DexMF presented an adequate size and appeared to be physiologically well tolerated as reported for dextrancoated-magnetite nanoparticle.6 Furthermore, it was efficient in mediating hyperthermia. Although the hyperthermia treatments induced by direct injection of magnetic iron oxide nanoparticles in the tumor are already in clinical phase I or II for certain kinds of tumors such as glioblastoma multiforme, prostate J. Biomed. Nanotechnol. 9, 1261–1271, 2013

carcinoma and pancreatic carcinoma,40–41 no similar study regarding magnetic iron oxide nanoparticles mediating hyperthermia associated or not with other therapies to treat patients with breast cancer (phase I or II studies) have been reported until now. For breast cancer, the use of magnetic nanoparticles has been mainly tested for detecting metastasis or targeting cells for tumor imaging, both in in vitro or animal model studies,42–45 and nanomedicine in treating breast cancer has been performed with drug delivery systems. Thus, while nanoparticles are also being used in in vitro research for gene therapy and targeted thermal ablation treatment of breast cancer, their role as drug carriers is the closest that nanooncology has come to the patient’s bedside.46 Hyperthermia using superparamagnetic iron oxide nanoparticles (SPIONs) for breast cancer in animal models seems not to have been tested until the present work. Additionally, the works already in clinical phase I or II used magnetite nanoparticles to induce hyperthermia, while SPIONs based on maghemite nanoparticles were used in this study. Maghemite is an oxidized form of magnetite and thus is chemically more stable.47 Ehrlich tumor is very aggressive and readily grows more quickly from the second week, reaching large sizes in short 1267

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periods. For ethical reasons, this fast growth impairs the sections showed viable tissue in all treated animals, the use of this tumor model for long periods due to the general necrotic area in the treated tumors continued to be stastate of the experimental animals. According to our pretistically higher than in non-treated tumors. In addition, vious report, 11 days after the subcutaneous implantation the ‘control’ tumor (tumor without treatment) significantly of Ehrlich tumor in mice may be considered too long, not increased its volume by over 200% from the first to the providing appropriate circumstances to investigate MHT second week, while the treated tumors had a non signifiefficacy.7 Additionally, under favorable conditions, such as cant increased in their volume from the first to the second those provided by some antioxidant interventions, Ehrlich week, and results of histology showed that the tumor voltumor readily forms metastasis.19 Thus, considering the ume was possibly affected by the infiltration of immune injection of 55 × 106 viable cells instead of previously cells and edema. Although in previous our report pequi reported 55 × 104 viable cells7 19 to produce a more clinoil alone reduced the tumor volume and increased the ically advanced tumor, one and two weeks were crucial necrotic area,19 considering the above discussion and the advanced clinical tumor now implanted, we can state that periods for investigation of these proposed treatments. the results were satisfactory in inhibiting tumor growth, Experimental studies support the role of ROS in the inimainly because magnetohyperthermia was conducted only tiation and promotion phases of carcinogenesis, in part by in the first 3 days after clinical tumor implantation and, showing that dietary antioxidants act as preventive agents after that, no treatment was performed. Thus, both theraof cancer.2 Previous results from Ehrlich-solid-tumorbearing mice corroborate this evidence, demonstrating that pies (MHT and PMHT) are promising and probably can antioxidant administrations of vitamins C and E or pequi enhance the inhibition of tumor growth by optimizing prooil before tumor inoculation were effective in inhibiting posed treatments. the growth of a less clinically advanced tumor.19 AdditionAlthough the accumulation of NPs in tumor tissues ally, MHT represents a novel and promising therapy for might occur by enhanced accumulation of the NPs in cancer treatment, by achieving the specific lysis of tumor tumors due to leaky tumor vasculature in combination cells, even in the absence of exposure to an alternate magwith poor lymphatic drainage,50 the tumor microvascula7 48 49 Thus, associating oral administration of ture has a discontinuous and loose nature,51 also favoring netic field. pequi oil before tumor inoculation with DexMF mediating the spread of the nanoparticles to the blood stream. Thus, magnetohyperthermia we could expect to reduce the carblood cells assayed can be used to evaluate the effects of Delivered by Publishing Technology to: Rice University cinogenic process and control IP: tumor growth, as observed. 188.112.177.181 On: Thu,the 04employed Feb 2016treatments. 20:56:59 Copyright: American Scientific Although clinical tumor volume was non-significantly GivenPublishers that antioxidant enzyme activities fall in a wide higher for both treatments compared to non-treated tumor variety of tumors,26–30 oxidative stress can occur, favoring tumor growth by increasing ROS generation. Because after the first week, results of tumor histology and morROS acts systemically, increasing the antioxidant funcphometry indicate that this increased volume was due to tion of the organism could prevent or retard this process. tissue edema. This edema may be related to the release Thus, although the DNA damage was measured only in of tumor necrosis debris and the associated inflammatory the peripheral leucocytes, this can be used to evaluate process induced in tumors submitted to the experimenthe antioxidant status of the whole organism and, consetal treatments. Although immunohistochemistry was not quently, to evaluate if this state had improved after treatused to validate claims of defensive cell infiltration, the ment. In this context, antioxidant administration with pequi increased number of neutrophils + monocytes observed for oil could help the antioxidant system, inhibiting tumor both treatments particularly after the first week corroborate growth. Although in this study a significant decrease in this suggestion. While Ehrlich tumor is very aggressive DNA damage occurred only for MHT at one week and this and spontaneously presents an intense necrosis process in significance disappeared at two weeks, PMHT treatment the central area,7 19 both treatments increased the necrosis percentage, and treated tumors differed from non-treated also decreased DNA damage compared to negative conby the presence of necrotic tissue in the peripheral areas. trol and non-treated tumor-bearing animals, one and two In these groups, necrotic areas were also highly infiltrated weeks after tumor inoculation. While this decrease was by defense cells, especially neutrophils, and there were statistically non-significant, it has biological importance, hemorrhagic regions with red blood cells outside the veins. because rapid tumor growth can induce increased ROS The infiltration of inflammatory cells was possibly also generation, which in turn can stimulate cell division and responsible for the non-significant reduction of tumor volcause damage to DNA, favoring faster progression of the ume, mainly because there was a significant increase in the carcinogenesis process.13 Moreover, after two weeks there was non-significantly increased DNA damage for the MHT percentage of necrotic area in the treated groups. Necrotic treatment. This was probably due to the use of dextran by tissue was also observed in the peripheral areas of treated the body, releasing iron from maghemite, which may have tumors and it is noteworthy that in two animals (33%) increased oxidative stress by Fenton reaction.12 In this way, treated with MHT no viable tumor tissue were detected the preventive pequi oil supplementation, even adminisin the histological examinations after the first week. Furtered only 10 days before tumor inoculation, seemed to be thermore, although after the second week the histological 1268

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protective, because PMHT therapy appeared to be more the Federal District (FAPDF), the Coordination for Further Training of Graduate Staff (CAPES), the CAPESeffective with the advance of the carcinogenesis process Rede CON-NANO, the NCT-Nanobiotecnologia, and the after the second week. CNANO-UnB for financial support. Dextran is a complex glucan polysaccharide used medicinally as an antithrombotic and as a volume expander.52 Thus, the non-significantly decreased RBC, REFERENCES HGB and HCT after the first week may have been due 1. H. S. Santos and W. M. S. Cruz, A terapia nutricional com vitaminas to an expansion of the plasma by diluting RBC and HGB antioxidantes e o tratamento quimioterápico oncológico. Rev. Bras. concentration. This suggestion is supported by results of Cancerol. 47, 303 (2001). 2. C. Borek, Dietary antioxidants and human cancer. Integr. 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Our findings evidence the potential use of sentes na polpa e na amêndoa do pequi (Caryocar brasiliense Camb.). DexMF mediating magnetohyperthermia in cancer treatRev. Bras. Frutic. 29, 695 (2007). 16. M. N. S. Oliveira, E. Gusmão, P. S. N. Lopes, M. O. M. Simões, ment and also suggest that the preventive pequi oil adminL. M. D. Ribeiro, and B. A. Souto, Estádio de maturação dos frutos e istration could increase the efficiency of this process. Acknowledgments: We are grateful to the Brazilian National Council for Technological and Scientific Development (CNPq), the Foundation to Support Research in J. Biomed. Nanotechnol. 9, 1261–1271, 2013

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Dextran-functionalized magnetic fluid mediating magnetohyperthermia combined with preventive antioxidant pequi-oil supplementation: potential use against cancer.

This work aimed to test a dextran-functionalized magnetic fluid (DexMF) sample in mediating magnetohyperthermia to treat an advanced clinical Ehrlich-...
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