Antitumor effect and toxicity of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles in mice bearing breast cancer
© Carneiro et al; licensee BioMed Central Ltd. 2013
Received: 2 October 2012
Accepted: 30 January 2013
Published: 16 February 2013
Magnetic fluids containing superparamagnetic iron oxide nanoparticles represent an attractive platform as nanocarriers in chemotherapy. Recently, we developed a formulation of maghemite nanoparticles coated with rhodium (II) citrate, which resulted in in vitro cytotoxicity enhanced up to 4.6 times when compared to free rhodium (II) citrate formulation on breast carcinoma cells. In this work, we evaluate the antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma.
Mice were evaluated with regard to the treatments’ toxicity through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine; DNA fragmentation and cell cycle of bone marrow cells; and liver, kidney and lung histology. In addition, the antitumor activity of rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate was verified by tumor volume reduction, histology and immunohistochemistry.
Regarding the treatments’ toxicity, no experimental groups had alterations in levels of serum ALT or creatinine, and this suggestion was corroborated by the histopathologic examination of liver and kidney of mice. Moreover, DNA fragmentation frequency of bone marrow cells was lower than 15% in all experimental groups. On the other hand, the complexes rhodium (II) citrate-functionalized maghemite and free rhodium (II) citrate led to a marked growth inhibition of tumor and decrease in CD31 and Ki-67 staining.
In summary, we demonstrated that both rhodium (II) citrate and maghemite nanoparticles coated with rhodium (II) citrate formulations exhibited antitumor effects against 4T1 metastatic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of maghemite nanoparticles coated with rhodium (II) citrate. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens its use for future breast cancer therapeutic applications.
Breast cancer represents the main cause of death in women worldwide due to the high metastatic capacity of this disease. Nowadays, chemotherapy is the most commonly used treatment in the therapeutic approach; however, conventional chemotherapy has shown low efficacy when the disease is not treated in early stages. As a matter of fact, chemotherapy drugs are not specific and act both on tumor and normal cells, causing side effects. Thus, the development of therapeutic strategies such as drug delivery systems (DDS) represents an area of great interest in cancer research[4–6].
Through the development of nanotechnology, nanomaterials designed to work as DDS are increasingly being used in nanomedicine. This field has advanced, presenting innovative approaches, allowing the improvement of cancer therapeutic treatments. Among various materials used for DDS, magnetic fluids containing superparamagnetic iron oxide nanoparticles (SPIOs) represent an attractive platform as nanocarriers in chemotherapy. SPIOs ranging from 10 to 500 nm in size can accumulate inside the interstitial space in tumors, since the blood vessel wall becomes more permeable than in the normal tissue state. This effect, added to the poor lymphatic drainage in tumor and the tumor microvasculature, which has a discontinuous and loose nature[9, 10], is known as enhanced permeability and retention (EPR) and is currently considered an effective way to bring drugs into tumors, especially drug-loaded nanocarriers. Thus, SPIOs have shown promising results in the field of oncology, acting as an efficient DDS for controlled drug release and penetration in solid tumors[4, 7, 12].
In the last decade, an increasing number of investigations using several types of iron oxide-based nanoparticles have been carried out. However, only maghemite (γ-Fe2O3) and magnetite (Fe3O4) are able to fulfill the necessary requirements for biomedical applications, due to their superior biocompatibility with respect to other magnetic materials. Nowadays, magnetic nanoparticles are being associated with several chemotherapy drugs, such as doxorubicin, docetaxel, methotrexate, tamoxifen, paclitaxel and cisplatin.
Recently, our group developed a formulation of maghemite nanoparticles (NPs) coated with citrate (Magh-cit) and rhodium (II) citrate (Magh-Rh2(H2cit)4), and we have shown that Magh-Rh2(H2cit)4 enhanced cytotoxicity on breast carcinoma cells by up to 4.6 times when compared to free rhodium (II) citrate (Rh2(H2cit)4). Likewise, we verified that Magh-Rh2(H2cit)4 induced higher in vitro cytotoxicity on breast carcinoma cells than on breast normal cells. Thus, in the present study, we aimed to evaluate the antitumor activity and systemic toxicity induced by intratumoral injections of Rh2(H2cit)4 and Magh-Rh2(H2cit)4 in Balb/c mice bearing 4T1 breast carcinoma.
Results and discussion
Systemic toxicity assessment in mice bearing 4T1 breast carcinoma
Effects of Rh 2 (H 2 cit) 4, Magh-Rh 2 (H 2 cit) 4 and Magh-citrate on hematology and biochemical parameters of female Balb/c mice 23 days after animals’ tumor transplantation 1
24555.00 ± 9693.69
45774.00 ± 11995.51
59060.00 ± 25390.07
10.07 ± 1.03
10.17 ± 0.86
14.75 ± 0.33
14.26 ± 0.14
13.36 ± 0.85
44.62 ± 0.96
44.40 ± 0.39
41.09 ± 2.51
38.50 ± 13.88
18.71 ± 7.66
0.50 ± 0.50
0.00 ± 0.00
1.43 ± 0.65
57.67 ± 13.40
84.00 ± 3.36
70.57 ± 7.40
0.00 ± 0.00
0.00 ± 0.00
0.14 ± 0.14
0.17 ± 0.17
0.00 ± 0.00
0.14 ± 0.14
3.17 ± 1.01
5.20 ± 1.11
9.00 ± 4.94
25.50 ± 3.57
20.86 ± 2.15
21.33 ± 3.38
0.10 ± 0.00
0.10 ± 0.00
0.13 ± 0.02
143.67 ± 14.22
127.57 ± 12.10
112.33 ± 15.23
Serum Fe (mg/dL)
Tumor volume regression and survival test
Citrate-functionalized-maghemite has been attested as providing successful nanoparticles in the production of biocompatible and stable magnetic fluids[30, 31]; however, our work was the first to investigate and demonstrate the antitumor effect of rhodium (II) citrate associated with nanoparticles on in vivo 4T1 orthotopic breast carcinoma model. Previous studies, conducted by Zyngier et al (1989), demonstrated that free rhodium (II) citrate has significant antitumor, cytotoxic, and cytostatic activity in Swiss mice bearing ectopic breast carcinoma model (Ehrlich cell line), which is in agreement with our results.
Nevertheless, two important aspects should be taken into consideration. First, unlike the study of Zyngier et al (1989), where the process of tumor implantation was ectopic, we transplanted tumor cells in the mammary gland (orthotopic implantation), which is the original environment of this cell line. This represents an important factor when developing therapies that are able to target not only the tumor but also its microenvironment components. In view of this, our results are of great importance for the study of the effects of anticancer drugs in the mammary microenvironment. Second, our study is the first to evaluate in vivo the effects of the association of rhodium (II) citrate to nanoparticles. We suppose that these associations could provide higher therapeutic efficacy than free formulations, since drug-loaded nanocarriers potentially provide controlled time release of the drug beyond modification of drug pharmacokinetics and biological distribution. Moreover, the interaction of nanoparticles with the biological environment is very important for designing strategies that should be selective at the pharmacological site, especially in the breast, where the blood flow and lymphatic drainage is intense.
Furthermore, we also evaluated the antitumor effect of Rh2(H2cit)4 and Magh-Rh2(H2cit)4 by the survival analysis of mouse groups, during the experimental period of 23 days. Deaths in the mouse control group (with untreated tumors) started on experimental day 16 after tumor implantation. On the 18th and 21st days, two deaths occurred in each group of animals treated with Rh2(H2cit)4 or Magh-citrate, and one death in the control group. All mice in the Magh-Rh2(H2cit)4 group survived until the end of the experimental period (23 days) (Figure4B). Magh-Rh2(H2cit)4 complex exhibited antitumor effect against 4T1 metastatic mouse breast carcinoma, without apparent systemic toxicity and a significant increase in treated mice survival period (period of 23 days). Zyngier and Kimura (1989) showed a percentage of 78.8% survival in Swiss mice treated with Rh2(H2cit)4 after implantation of Ehrlich tumor cells, in a period ranging from 23 to 27 days. These findings are consistent with the present study since we observed the same survival percentages in Balb/c mice treated with free Rh2(H2cit)4 (about 75%), after 4T1 cell implantation for a similar period of time (Figure4B).
In our previous study, we reported that Rh2(H2cit)4 induced cytotoxicity on both breast cancer and breast normal cells, in vitro. However, rhodium associated with the nanoparticles (Magh-Rh2(H2cit)4) was more cytotoxic to breast cancer cell lines. These findings are consistent with the present in vivo study since mice treated with Magh-Rh2(H2cit)4 showed a decrease in tumor volume, without occurrence of deaths or macro and microscopic alterations.
Although rhodium (II) citrate-coated maghemite nanoparticles have never been described before, another study reported the association of rhodium (II) citrate with hydroxypropyl-beta-cyclodextrin, macrocyclic oligosugars from biodegradable polymer. In this study, the association with these oligosugars led to the minimization of nonspecific toxicity since they increased the efficiency of encapsulation and the duration of rhodium (II) citrate release.
On the other hand, some strategies have been used to increase the specificity of antitumor agents by associating drugs with nanocarriers. SPIOs, for instance, are considered one of the most important nanocarriers due to their inherently low particle size, high magnetization values and ability to load anticancer agents, allowing enhanced therapeutic selectivity provided by local hyperthermia, magnetic targeting and magnetic resonance imaging[36, 37]. Furthermore, SPIOs are small enough to escape renal clearance and opsonization processes, thus being able to accumulate easily in the tumor. The Magh-Rh2(H2cit)4 complex used in this study contains nanoparticles with sizes of 10 nm thus being small enough to escape renal clearance and opsonization processes, which can promote a preferential accumulation in the tumor due to its abnormal vascular nature. Hence, the nano-sized property was exploited to take advantage of alterations that occur in tumor cells and adjacent tissues.
Histological analyses showed that the 4T1 tumor model without treatment was invasive and characterized by typical and atypical mitoses, poor differentiation, moderate pleomorphism with neovascularization areas and infiltration of inflammatory cells in tissue (data not shown). Previous reports showed that 4T1 tumor cells display a multistage tumor development pattern with atypical hyperplasia, corroborating our findings. The 4T1 orthopic breast tumor is a model that shares similarities with metastatic human breast cancer, thus representing a good model to evaluate the efficacy of anticancer drugs.
Regarding Rh2(H2cit)4 and Magh-Rh2(H2cit)4 treatments, we observed intense necrosis and fibrosis, demonstrating that its antitumor effectiveness can be associated with cell death triggered in tumor tissue (Figure5F-H). Moreover, in mice treated with Rh2(H2cit)4 we observed inflammatory edema (Figure5E), as well as the presence of nuclear fragments observed in Rh2(H2cit)4 and Magh-Rh2(H2cit)4 groups, which may be related to tumor cell death by apoptosis (Figure5D, I). These results corroborate the tumor regression observed in mice treated with these compositions.
CD31 and Ki-67 immunohistochemistry analysis
High levels of CD31 (angiogenesis marker) are correlated with poor prognosis and decreased survival of breast cancer patients. Studies demonstrated that during prostate cancer treatment with cryoablation a positive correlation between necrosis and hypoxia and negative correlation with microvessel density was observed through the decrease in CD31 expression and the increase in necrosis area. Further, another study demonstrated that tumor growth delay is associated with decrease in microvessel density and increase in tumor cell apoptosis. Thus, the inhibition of tumor angiogenic factors is important since they are correlated with the metastasis process and cell death.
Histopathological analysis showed numerous necrosis areas and low Ki-67 and CD31 expression in 4T1 tumor tissues treated with Rh2(H2cit)4 and Magh-Rh2(H2cit)4. Therefore, we suggest that the antitumor efficacy of these formulations is correlated with a reduction in Ki-67 and CD31 molecules, which indicates a decrease in both the cellular proliferation and microvessel network development. Overall, the histopathology, immunohistochemistry and regression tumor findings are consistent and linked.
In summary, we demonstrated that both Rh2(H2cit)4 and Magh-Rh2(H2cit)4 formulations exhibited antitumor effects against 4T1 orthotopic breast cancer cell line following intratumoral administration. This antitumor effect was followed by inhibition of both cell proliferation and microvascularization and by tumor tissue injury characterized as necrosis and fibrosis. Remarkably, this is the first published report demonstrating the therapeutic efficacy of Magh-Rh2(H2cit)4. This treatment prolonged the survival period of treated mice without inducing apparent systemic toxicity, which strengthens the case for its use in future breast cancer therapeutic applications.
Preparation of the rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles
The maghemite nanoparticles (Magh-citrate) and rhodium citrate, Rh2(H2cit), as well as the rhodium(II) citrate-loaded superparamagnetic iron oxide nanoparticles (Magh-Rh2(H2cit)4), (Figure1) were prepared and characterized as previously described. Briefly, Rh2(H2cit)4 was synthesised by exchange trifluoroacetate ligands from the precursor rhodium(II) trifluoroacetate by citrate ligands. The compound was obtained as a green aqueous solution with a standardised concentration of 0.054 mol L-1. Maghemite nanoparticles were synthesised by alkaline co-precipitation of Fe2+ and Fe3+ ions. The particles obtained in the magnetite (Fe3O4) phase were oxidised to maghemite (γ-Fe2O3) by bubbling of oxygen gas and were subsequently purified by dialysis with deionised water for several days ([Fe] = 0.37 M). The Magh-Rh2(H2cit)4 was prepared using 5 mL of the colloidal dispersion with 1 mL of Rh2(H2cit)4 and stirred for 24 hours.
Orthotopic tumor cell implantation and treatment
Balb/c female mice (12 weeks old) were purchased from Cemib-UNICAMP (São Paulo, Brazil). All mice were maintained in plastic cages under standard conditions of 12 h dark/light cycle. The mice, weighing 20-25 g, were fed with standard diet and water ad libitum. All experiments described were approved by the Animal Research Ethics Committee of the University of Brasilia - Institute of Biologic Sciences, Brazil.
The 4T1 breast carcinoma cells were thawed and cultivated in flasks with Dulbecco’s modified eagle’s medium (DMEM) supplemented with 1% penicillin and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere 5% CO2. Two weeks later, Balb/c mice were anesthetized with ketamine (80 mg/kg) and xilazin (10 mg/kg) via intraperitoneal. Then, 2×104 4T1 cells (in suspension in 50 μL serum-free DMEM) were injected (1 mL-gauge needle) in their mammary gland, which is the natural primary microenvironment of breast tumor occurrence. Seven days after implantation of 4T1 cells, mice were divided into four groups (n=8/group), and each group was treated with 50 μL of (1) Rh2(H2cit)4, (2) Magh-Rh2(H2cit)4, (3) Magh-citrate or (4) water. Peritumoral injections were carried out every three days, totalizing seven applications of 0.3 mg/kg rhodium (II) citrate (total dose of Rh2(H2cit)4 was 2 mg/kg).
Mice treated with Magh-citrate received the same iron concentration and nanoparticle amount found in Magh-Rh2(H2cit)4 (0.37 M and 2.4×1015 particles). Animals without tumor and without treatment (healthy) were also included in this study as control groups. On the 23rd experimental day after tumor implantation, the mice of each experimental group were euthanized and the tumor, liver, kidney, and lung were collected to perform antitumor and systemic toxicity analysis as described in items 2.4, 2.5 and 2.6.
Systemic toxicity assessment in mice bearing 4T1 breast cancer
In order to evaluate potential systemic toxicity induced by the treatments, analyses of macroscopic aspects, histology, blood, DNA fragmentation and cell cycle were performed. Treated mice were continuously monitored for relevant indexes such as weight loss, diarrhea, skin ulcers and deaths.
Before euthanasia, which was by cervical dislocation, animals were anesthetized with the mixture of xylazine and ketamine described previously (item 2.3). Blood samples (1 mL/animal) collected by cardiac puncture were used to carry out hemogram and biochemical dosages of serum alanine aminotransferase (ALT), iron and creatinine. Hemogram was processed in a multiple automated hematology analyzer (XZ 2100 Sysmex equipment) and serum biochemical analyses were run on the automated chemistry analyzer ADVIA 2400 (Siemens), using the appropriate Advia chemistry reagents, protocols and controls.
After euthanasia, bone marrow (BM) cells were collected from femurs and resuspended in one milliliter of fetal bovine serum (FBS, Gibco) to perform DNA fragmentation and cell cycle analyses, which is a rapid detection method of chromosome damage and interference with cell mitosis caused by several agents. Cells were fixed in cold 70% ethanol, and stored overnight at -30°C. The cells were centrifuged and incubated with 300 μL of lysis buffer (0.1% sodium citrate, 0.1% Triton X-100 and 20 μg/mL of propidium iodide, diluted in PBS pH 7.4) for 30 min at room temperature and protected from light. DNA fragmentation and the cell cycle were analyzed using FACS Calibur flow cytometry (Becton & Dickenson, USA) and a total of 10,000 events were collected per sample. Histopathology analysis of the liver, kidneys and lungs was also performed in order to verify possible toxic effects induced by treatments.
Tumor regression and survival analysis
To evaluate tumor regression, tumors were surgically removed, their width and length measured by a digital pachymeter (Stainless, hardened), and their respective volumes calculated according to the formula of Yanase et al. (1998): length × width2 × 0.52. Animals that had died were submitted to necropsy, and the time of their death was recorded.
Histopathology and immunohistochemistry analysis
Tumors were fixed in 10% phosphate-buffered formalin overnight (room temperature), 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) or Perls Prussian Blue for histological analyses (light microscopy). Histological sections were examined to verify the presence of nanoparticles, cell proliferation pattern, pleomorphism, degree of cell differentiation and cell death.
Immunohistochemical analyses were performed in order to analyze cell proliferation by Ki-67 staining and vascularization by CD31 staining in the tissues. After paraffin removal and hydration, histological sections were immersed in citrate buffer (3 mM, pH 6.0) for 10 minutes at 120°C for antigen retrieval. Subsequently, non-specific binding sites were blocked with 3% normal serum or BSA. Afterward, the sections were incubated with anti-Ki-67 (1:200 Abcam, ab15580) or CD31 antibodies (1:200 Dako, K4067, Glostrup, Denmark) for 2 h at room temperature, washed and then incubated with biotinylated secondary antibodies for 20 min followed by avidin-biotin complex (LSAB-HRP Kit, Dako, K0690, Glostrup, Denmark). After washing, sections were incubated with diaminobenzidine substrate and counterstained with Mayer's hematoxylin.
All cells were counted in five consecutive microscopic high power fields (400x) using an integration graticule (CARL ZEISS-4740680000000-Netzmikrometer 12.5×). At this magnification, each field has an area equal to 0.015625 mm2; thus a total area equal to 0.078125 mm2 was analyzed in each specimen.
To evaluate differences in tumor volume in each experimental group, after the treatments in Balb/c mice bearing 4T1 breast carcinoma, the following tests were run: inhibition of cell proliferation (by quantification of positive Ki67 staining cells number) in 4T1 breast carcinoma tissue; levels of serum ALT, creatinine and iron and peripheral blood counts (in order to verify the systemic effects of the treatments with Rh2(H2cit)4 and Magh-Rh2(H2cit)4); and in the proportion of DNA fragmentation or the cell cycle phase number of bone marrow of mice, the one-way Analysis of variance (ANOVA) was performed. When statistically significant differences were found, analysis was complemented by the Tukey test or the Bonferroni method. Before the intragroup comparison, the Shapiro Wilk test was conducted, to check whether each variable was normally distributed. Between-group comparisons of weight on different days after tumor transplantation were performed using a two-way ANOVA with post-hoc Dunnet test.
To describe the survival prolongation effect after the different treatments in Balb/c mice bearing 4T1 breast carcinoma, the Kaplan-Meier statistical method was used to generate survival curves. Then, these survival curves were compared using the Mantel-Haenszel log-rank test.
Data were presented as means ± SE. The significant level adopted was 5%. Calculations were done using the SPSS, Inc., Chicago, IL software (version 17.0). All plots were generated using GraphPad Prism 5.0 (GraphPad Software, La Jolla, Calif.).
Marcella Lemos Brettas Carneiro is the first author of this work.
This research was supported by the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPQ), “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), “Fundação de Apoio a Pesquisa no Distrito Federal” (FAP-DF, Grant: 193.000.466/08), “Financiadora de Estudos e Projetos” (Finep) and the “Instituto Nacional de Ciência e Tecnologia (INCT) em Nanobiotecnologia”. The authors are grateful to SABIN Laboratory for technical support in hematologic analysis and to Viviane Montanari Leal for technical support in immunohistochemistry procedures.
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