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), (Figure
1) 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.).