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British Journal of Radiology (2004) 77, 573-580
© 2004 British Institute of Radiology
doi: 10.1259/bjr/71404908
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Full Paper

Synthesis of copper octabromotetracarboranylphenylporphyrin for boron neutron capture therapy and its toxicity and biodistribution in tumour-bearing mice

M Miura, PhD G M Morris, PhD P L Micca, BS M M Nawrocky, BA M S Makar, BA S P Cook, BA and D N Slatkin, MD

Medical Department, Brookhaven National Laboratory, Upton, New York 11973-5000, USA


   Abstract
Top
 Abstract
Introduction
 Methods and materials
 Results
 Discussion
 Conclusion
 References
 
Copper tetracarboranyltetraphenylporphyrin (CuTCPH) is a minimally toxic carborane-containing porphyrin that has safely delivered high concentrations of boron for experimental boron neutron capture therapy (BNCT). Copper octabromotetracarboranylphenylporphyrin (CuTCPBr), synthesized by bromination of CuTCPH, is one of several new minimally toxic analogues of CuTCPH being studied in our laboratory, which could possess comparable or better tumour-targeting properties with enhanced tumour cytotoxicity. Its biodistribution, biokinetics and toxicity in mice with subcutaneous EMT-6 (mammary) or SCCVII (squamous cell) carcinomas were compared with those of CuTCPH. The administration of ~200 mg kg–1 of either porphyrin in six intraperitoneal injections over 2 days had no apparent effect, but administration of ~400 mg kg–1 slightly lowered body weights, elevated alanine and aspartate transaminase activities in blood plasma, and depressed blood platelet counts for several days. Enzymes and platelets returned to normal within 5 days after those injections and body weights returned to normal within 2 weeks. High average concentrations of boron from either porphyrin were achieved in the two tumour models from a total dose of ~200 mg kg–1. The high tumour boron concentration decreased slowly while concentrations in blood decreased rapidly. Boron concentrations in brain and skin were consistently lower than in tumour by a factor of 10 or more. Although either CuTCPH or CuTCPBr can be labelled with 64Cu for imaging by positron emission tomography (PET), CuTCPBr can also be labelled by 76Br, another PET-imageable nuclide.


   Introduction
Top
 Abstract
 Introduction
Methods and materials
 Results
 Discussion
 Conclusion
 References
 
A number of novel carboranylporphyrins have been evaluated in mice as boron delivery agents for possible use in boron neutron capture therapy (BNCT), a bimodal technique based on the selective delivery of 10B to a tumour by boron-containing carrier molecules and subsequent irradiation with slow neutrons [1]. The short-range (5–9 µm), high linear-energy-transfer (LET) alpha and lithium-7 ionizing particles produced by the resultant 10B(n, {alpha})7Li nuclear reactions can palliate or ablate 10B-rich tumours. A sufficiently low concentration of 10B in normal tissues should spare the latter from serious radiotoxicity [2]. The compounds currently used for clinical trials of BNCT of malignant brain tumours are the sulphydryl boron hydride Na2B12H11SH (BSH) and the amino acid p-boronophenylalanine (BPA), which yield tumour:blood boron concentration ratios of 1:1 and 3:1, respectively [3–6]. It would be useful if combinations of these and/or new boron carriers could achieve higher ratios without undue chemotoxicity.

Photodynamic therapy (PDT) is a clinically established bimodal technique of cancer therapy whereby a superficial tumour loaded with a photosensitizing porphyrin or related macrocycle is irradiated with red light, which catalyzes the conversion of molecular oxygen to chemically reactive species [7, 8]. These species diffuse rapidly and initiate cytotoxic chain reactions in cell membranes, especially those of the well-oxygenated zones of tumour. A potential advantage of BNCT over PDT is that, whereas in PDT red light penetrates only several millimetres in tissues, in BNCT epithermal neutrons penetrate effectively to depths of 5–7 cm. Another distinguishing aspect of BNCT is that the energetic 7Li and alpha particles formed by the neutron-10B reaction do not require oxygen to be optimally radiotoxic, and therefore can sterilize quiescent malignant cells in poorly oxygenated parts of a tumour.

Remarkably, tumour:blood boron concentration ratios steadily increased to over 100:1 for several days after injection of lipophilic boron-containing porphyrins [9–11], in contrast to tumour:blood boron concentration ratios from BSH or BPA, which remain constant after injection with tumour retention times of only a few hours [12, 13]. The only boronated porphyrin that has been shown to be efficacious in vivo for BNCT of any experimental tumour is the carborane-containing porphyrin copper tetracarboranyltetraphenylporphyrin (CuTCPH) [11].

Herein we describe the synthesis of the octabromo analogue of CuTCPH, namely copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetra(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin, (CuTCPBr, 21.7 wt% B) shown in Figure 1Go. Also described are the biodistribution and toxicity of CuTCPBr, as well as comparisons with those of CuTCPH and BPA. For treatment planning purposes, both CuTCPH and CuTCPBr could be labelled by 64Cu, a positron-emitter with a half-life of 12.7 h, and imaged non-invasively in tumours and normal tissues for BNCT by positron emission tomography (PET). CuTCPBr provides more flexibility in that PET imaging could also be implemented by using 76Br, a positron-emitter with a longer half-life of 16.2 h [14]. The animal tumour models used for the biodistribution and toxicity studies reported here were the murine EMT-6 mammary [15] and SCCVII squamous cell carcinoma models [16].



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  		Figure 1. Structures of CuTCPBr, its precursor CuTCPH and its congener CuTCP.

 

   Methods and materials
Top
 Abstract
 Introduction
 Methods and materials
Results
 Discussion
 Conclusion
 References
 
Synthesis of CuTCPBr
All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Mallinkrodt, Inc (Phillipsberg, NJ). Fast atom bombardment (FAB) mass spectra were carried out by the State University of New York at Stony Brook Mass Spectrometer Facility. Optical spectra were obtained with a Carey 50 Spectrophotometer (Varian Inc., Palo Alto, CA) and FT-IR was carried out on a Mattson Polaris spectrometer (Thermo Mattson, Madison, WI). CuTCPH [17] (200 mg, 0.15 mmol), dissolved in 1:1 CHCl3/CCl4 (CC) (40 ml), was brominated by addition of bromine (240 µl, 4.6 mmol) in CC (10 ml), which was added dropwise over a period of ~30 min while stirring [18]. The solution was stirred at room temperature for ~4 h, at which time pyridine (0.6 ml) in CC (8 ml) was added over 30 min and allowed to stir for ~16 h. The reaction was monitored using optical spectroscopy and thin-layer chromatography (TLC).

Sodium metabisulphite (20% aqueous, 40 ml) was used to quench the excess bromine. After the product was extracted using dichloromethane the organic layer was washed three times with water, and dried (anhydrous sodium sulphate), the solvents were removed in vacuo. CuTCPBr was purified by preparative TLC (alumina) using 85:15 hexane:ethyl acetate as eluent. The yield was 176 mg (59%). UV-Vis absorption {lambda}max (nm) {varepsilon} acetone: 363 (20 400), 468 (90 100), 585 (12 500) FT-IR (KBr) 2582 cm–1 (BH); FAB Mass spectrum (NBA): m/z calc 1996; found 1996 Elemental Analyses: Calc. for C56H68N4O4B40Br8Cu; C 33.69, H 3.43, N 2.81; found C33.65, H 3.62, N 2.43.

To prepare a solution of ~3.3 mg ml–1 in 9% Cremophor EL (CRM) and 18% propylene glycol (PRG), the porphyrin was dissolved in tetrahydrofuran (THF) (1.5% of the total volume) and then heated to 40°C for 15 min [17]. CRM (9% of total volume) was then added and the mixture was heated to 60°C for 2 h, which removed most of the THF. After cooling to room temperature, PRG (18% of total volume) was added, followed by slow dropwise addition of saline (71.5% of total volume) with rapid stirring. The solution was degassed by stirring under vacuum (~30 mm Hg) for 30–60 min and then filtered (Millipore, 8 µm).

Animal models
All in vivo experiments were approved by the Brookhaven National Laboratory (BNL) Institutional Animal Care and Use Committee. Mice were purchased from Taconic Farms (Germantown, NY). The animal tumour models used for the biodistribution and toxicity studies reported here were the EMT-6 mammary [15] and SCCVII squamous cell [16] carcinomas in mice. Female BALB/c mice (18–22 g) were used for EMT-6 biodistribution studies and female C3H mice (20–25 g) were used for SCCVII biodistribution studies.

EMT-6 tumour model
To initiate subcutaneous (s.c.) EMT-6 tumours on the dorsal thorax, BALB/c mice were implanted s.c. with single-cell suspensions of 2.5 x 105 cells in 0.05–0.10 ml of culture medium. EMT-6 tumour cells were grown in vivo and in vitro in succession [15]. Single-cell monolayers were prepared from mouse-grown tumours by trypsinization, expanded in alpha MEM with 10% fetal bovine serum (FBS) for several passages. Aliquots of the cells in 10% DMSO were frozen in liquid nitrogen for storage. Prior to implantation in mice, an aliquot of the cells was thawed and regrown in tissue culture. Porphyrin injections were initiated 11 days after tumour cell implantation.

SCCVII tumour model
SCCVII murine squamous cell carcinoma cells were implanted s.c. in the dorsal thorax of C3H mice with 5 x 105 cells in 0.1 ml [16]. SCCVII cells were kindly provided by Professor J Martin Brown at Stanford University. They were grown in culture in D-MEM enriched with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine. Only passages 1–3 are used for tumour initiation. BPA or porphyrin injections were initiated 11 days after tumour implantation.

Compound administration
Tumour-bearing mice were given multiple i.p. injections of porphyrin (~200 or ~400 mg kg–1 porphyrin). BALB/c mice with EMT-6 tumours and C3H mice with SCCVII tumours were given total doses of ~200–400 mg kg–1 CuTCPBr in 6 i.p. injections over 2 days (3 per day at 4 h intervals). Injections were initiated 11 days after tumour implantation, when tumour volumes reached ~100–200 mm3. For the ~200 mg kg–1 CuTCPBr dose, a volume of 0.01 ml (g body weight)–1 per injection was used. For the ~400 mg kg–1 CuTCPBr dose, a volume of 0.02 ml (g body weight)–1 per injection was used. For the pharmacokinetic study, mice were euthanized 2, 4, or 6 days after the last injection. Tumour, blood, brain, liver, and pinna (mostly skin) samples were assayed for boron. For the 2-day biodistribution study in the EMT-6 tumour model, mice were euthanized 2 days after the last injection of CuTCPH or CuTCPBr and various tissues were sampled for boron assay.

Normal mice were given multiple i.p. injections of ~400 mg kg–1 porphyrin. Three groups of six BALB/c mice, without implanted tumours, were serially i.p.-injected with ~400 mg kg–1 using the protocol described above. A similar group of 3–6 mice were given excipient only. Mice were euthanized 2 days, 7 days, or 4 months after the last injection. Blood was analysed for haematological and enzymatic parameters for indications of toxicity.

Tumour-bearing mice were given single i.p. injection of BPA: Three groups of 5 tumour-bearing mice (SCCVII or EMT-6) were given a single i.p. injection of a 79 mg ml–1 of a BPA:fructose 1.0:1.1 molar solution (790 mg kg–1; 38 mg B kg–1). Mice were euthanized 1, 3, or 6 h after the injection. Tumour, blood, brain and liver were assayed for boron.

Boron analyses
Direct current plasma-atomic emission spectroscopy (DCP-AES) (ARL/Fisons Model SS-7) was used to assay boron (detection limit: 0.1 µg B ml–1) [19, 20]. Samples from individual mice (50–130 mg) were digested at 60°C with sulphuric acid:nitric acid (1:1). Triton X-100 and water were added to give final concentrations of ~50 mg tissue ml–1, 15% total acid v/v and 5% Triton X-100 v/v. Tissue samples were analysed from individual mice. Boron concentrations of injection solutions were determined by prompt-gamma spectroscopy [21], which was carried out at the Massachusetts Institute of Technology Reactor Prompt-Gamma Neutron Activation Facility.

Blood analyses
Blood was analysed for haematological and enzymatic indicators of toxicity. Haematologic assays are carried out at BNL using a VetScan HMT Hematology Analyzer, (Abaxis, Sunnyvale, CA) and enzymatic assays were performed by AniLytics, Inc. (Gaithersburg, MD). Concomitant assays of blood plasma, to which CuTCPBr was added, were carried out in vitro to ensure that the porphyrin itself does not interfere with enzyme assays. Mice were weighed daily and necropsies were carried out promptly after euthanasia by experienced observers (MMN, MSM, PLM).


   Results
Top
 Abstract
 Introduction
 Methods and materials
 Results
Discussion
 Conclusion
 References
 
Biodistributions in mice
The pharmacokinetics of boron in tumour, blood, and brain from mice given 215–230 mg kg–1 CuTCPBr (47–50 mg B kg–1) at 2, 4, and 6 days after the last injection are compared in Figure 2Go. Blood and brain boron concentrations were negligible in both strains of mice at all three time points. The porphyrin appears to show a somewhat greater affinity for the EMT-6 carcinoma than for the SCCVII carcinoma, particularly at the 4- and 6-day time points. This is mainly attributable to the longer retention time in EMT-6 than in the SCCVII carcinoma. However, at 2 days after the last injection, the tumour:blood and tumour:brain boron ratios were greater for the SCCVII tumour at ~120:1 and ~700:1, respectively, than those for the EMT-6 tumour at ~20:1 and ~125:1, respectively.



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  		Figure 2. Boron concentrations (µg B per g wet tissue) (mean±SD) in tumour, blood and brain at various time points (2, 4 or 6 days after last injection) after a dose of 215–30 mg kg–1 CuTCPBr (47–50 mg B kg–1) (n=7) in 6 i.p. injections over 2 days (a) in BALB/c mice bearing EMT-6 carcinomas and (b) in C3H mice bearing SCCVII carcinomas.

 
The boron concentrations in the tissues shown in the first two columns of Table 1Go indicate that the biodistribution properties of CuTCPBr are similar to those of CuTCPH in BALB/c mice bearing EMT-6 tumours despite the ~25% lower boron dose of CuTCPBr. When the CuTCPBr dose was doubled, boron levels in tissues were likewise doubled by day 2, except those in blood, which had increased from 0.4 to 35 µg g–1. However, after 2 more days those boron levels in blood decreased to less than 1 µg g–1 and levels in other normal tissues also decreased, but to a lesser degree. Figure 2aGo shows that the EMT-6 tumour boron level at day 6 had only decreased 25% from its 2-day value and the tumour:blood boron ratio had increased from 6.7:1 to >200:1.


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  		Table 1. Boron concentrations (µg B per g wet tissue) in various tissues from mice after administration of CuTCPH or CuTCPBr. All values are indicated as median (range)

 
BPA, as expected, had a brief pharmacokinetic clearance profile in comparison with CuTCPBr in both the EMT-6 and SCCVII carcinoma models (Figure 3Go). However, both tumours show good accumulation of boron from BPA, particularly at the 1-h time-point. At that time the EMT-6 tumour showed tumour:blood and tumour:brain boron ratios of 2.1 and 5.4, respectively, and the SCCVII tumour showed tumour:blood and tumour: brain boron ratios of 2.5 and 4.8, respectively. BPA, in contrast to CuTCPBr, shows a greater affinity for the SCCVII than for the EMT-6 carcinoma.



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  		Figure 3. Boron concentrations (µg B per g wet tissue) (mean±SD) in tumour, blood and brain at various time points (1, 3 or 6 h after a single i.p. injection) after a dose of ~790 mg kg–1 BPA fructose complex (~38 mg B kg–1) in (a) in BALB/c mice bearing EMT-6 carcinomas and (b) in C3H mice bearing SCCVII carcinomas.

 
Chemotoxicity
Mice given ~200 mg kg–1 CuTCPBr and euthanized 2, 4, or 6 days after the last of 6 injections exhibited no significant weight loss in comparison with controls. Nearly all haematologic parameters (Table 2Go) were comparable with those of similar mice given solvent only. However, when the dose was doubled to ~400 mg kg–1, slight lethargy and weight loss at days 2 and 7 were noted for CuTCPBr (Table 3Go). At necropsy, all major organs including the thymus and adrenal glands appeared normal except for a faint to distinct porphyrin-related greenish hue. The fecal contents of the colon were normal at necropsy and brown staining of perianal fur was not seen, which indicated that there had been no diarrhoea in vivo.


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  		Table 2. Haematological parameters from the whole blood of mice given ~200 mg kg–1 CuTCPBr in 9% Cremophor EL (CRM) given in 6 i.p. injections over 2 days or given 9% CRM in 6 i.p. injections over 2 days. All values are indicated as median (range)

 

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  		Table 3. Haematological and enzymatic parameters from the whole blood of (1) control mice (i.e. age-matched mice that were uninjected or given 9% CRM in 6 i.p. injections over 2 days), (2) mice given ~400 mg kg–1 body weight CuTCPBr in 9% CRM given in 6 i.p. injections over 2 days and (3) mice given ~400 mg kg–1 body weight CuTCPH in 9% CRM given in 6 i.p. injections over 2 days. All values are indicated as median (range)

 
Compared with mice that received Cremophor only and to age-matched control mice, some alterations in blood platelet counts and in plasma alanine and aspartate transaminase levels were observed at day 2 in mice that had received the higher ~400 mg kg–1 CuTCPBr dose (Table 3Go). However, 5 days later, those effects were no longer detectable. Other parameters tested (red blood cell counts; leukocyte counts, blood plasma creatinine, alkaline phosphatase and glucose levels) were consistently normal (data not shown). It was determined that CuTCPBr added to blood plasma in vitro did not interfere with plasma enzyme assays. Similar doses of CuTCPH and CuTCPBr induced comparable transitory reductions in blood platelets.


   Discussion
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
Conclusion
 References
 
Radiotherapy remains the most widely used treatment in the management of cancer. However, normal tissue morbidity limits its efficacy. Nevertheless, some progress has been made in improving radiotherapy by administration of chemotherapeutic and/or radiation sensitizing agents with preferential affinity to tumours before irradiation. There is growing interest in using porphyrins and/or porphyrin-related compounds to target tumours for BNCT [22–24]. CuTCPBr is currently undergoing pre-clinical evaluation as a tumour-targeting boron carrier in mice and rats.

Chemotoxicity has been encountered in previous attempts to develop porphyrins as boron delivery agents for BNCT [25–27]. The daunting hurdle to be surmounted was that typical doses required for BNCT, 200–400 mg kg–1 of porphyrin, are many orders of magnitude higher than those required for PDT, which requires only 0.2–1.0 mg kg–1 of porphyrin or other macrocycle. Porphyrin chemotoxicity is therefore not readily observed in PDT. Phototoxicity is the principle side-effect of PDT, for even low concentrations of a light-responsive porphyrin in areas of skin inadvertently exposed to sunlight can cause severe damage. The copper porphyrins used in our BNCT studies [11] are not responsive to light and therefore, are neither suitable for PDT nor likely to damage the skin. Serious chemotoxicity has never been observed during our studies of CuTCPH given to mice in therapeutically effective doses of up to 400 mg kg–1 [11, 17]. CuTCPBr has also been found non-toxic at such doses. Moreover, mice given up to 400 mg kg–1 of either CuTCPBr or CuTCPH showed only minor abnormalities in a few indices of toxicity 2 days after the last injection. However, these effects were not associated with the overt debility and mortality observed in some mice given half those doses of any of the water-soluble porphyrins [25–27].

CuTCPBr was synthesized in the course of a program to design and test analogues of CuTCPH that possess comparable or better tumour-targeting characteristics with enhanced tumour cytotoxicity. The biodistribution properties of CuTCPH in the murine EMT-6 tumour model have been reported previously [17] and used herein for comparison with those of CuTCPBr. The higher median blood boron concentration of 35 µg g–1 from the 430 mg kg–1 dose of CuTCPBr compared with 0.4 µg g–1 from the 215 mg kg–1 dose may reflect rapid saturation of a large tissue (most likely liver) reservoir of porphyrin by the much greater dose of porphyrin injected, thereby greatly slowing the clearance rate of the porphyrin from the blood.

For treatment planning purposes, CuTCPBr has an advantage over CuTCPH because both 76Br (half-life 16.2 h) and 64Cu (half-life 12.7 h) can be used for localizing the former by PET imaging. However, 64Cu can be produced at higher specific activity; e.g. at levels ~10 mCi positron activity per nanomole of Cu [28], compared with ~0.5 mCi positron activity per nanomole of Br [29]. Although there are 8 bromine atoms and only 1 copper atom per molecule of CuTCPBr, the initial advantage would be about 2.5 to 1 (~10/0.5)/8 in favour of 64Cu with regard to the specific activities noted above. However, by 78 h (solving for t in: 2.5 exp [–t.ln2/12.7]=exp [–t.ln2/16.2]) after comparable radiolabelling, an initially greater specific activity of 64Cu-CuTCPBr would equalize with that of the longer half-life of 76Br-CuTCPBr and could provide the latter tracer a progressively greater advantage for a multiple-day biokinetic study of a CuTCPBr-infused patient.

To our knowledge, CuTCPH is the only porphyrin shown to have palliated or ablated a malignant tumour in vivo using BNCT alone [11]. Because CuTCPBr's delivery of 10B to the EMT-6 tumour is similar to that of CuTCPH, the two compounds should be equally effective for BNCT. Furthermore, human head and neck malignancies, of which the vast majority (~80%) are squamous cell carcinomas [30] and have long been known to resist effective treatment by fractionated photon radiotherapy [31], may be more responsive to porphyrin-enhanced BNCT.

Although the boron concentrations in the SCCVII carcinoma from a porphyrin dose of 215 mg kg–1 (47 mg B kg–1) were generally less than those in the EMT-6 carcinoma, median tumour boron concentrations of 74 µg g–1 and tumour:blood boron ratios greater than 100:1 were attained. The SCCVII tumour model, used widely to study the palliative effects of chemotherapeutic drugs and/or radiotherapies [32, 33], should be of general interest in research of porphyrin-augmented BNCT. Thus, the outcome of combined BPA- and CuTCPBr-mediated BNCT of the SCCVII tumour may be predictive of the potential efficacy of clinical BNCT for intractable squamous cell carcinomas.

Given the relatively short retention time of BPA in tumours and the constant tumour:blood and tumour:brain boron ratios of ≤3:1 over time, CuTCPH or CuTCPBr could considerably enhance BPA-mediated BNCT. This is due to the substantially greater boron concentration and longer retention time in tumours of these porphyrins and their consistently low concentrations in blood and contiguous normal tissues. The BPA fructose alone delivered mean boron concentrations of 37–40 µg g–1 to SCCVII and EMT-6 tumours 1 h after injection, which may be significant enough to prove therapeutically effective. Similar kinetics were also observed in murine subcutaneous B16 melanomas [34]. Recent studies of BPA in rats bearing intracranial brain tumours have shown that the microlocalization of BPA in the main tumour mass and in tumour cells infiltrating normal brain can be significantly improved by long-term infusion and blood–brain barrier disruption [35, 36]. Thus such improvements in the delivery of BPA combined with the results of the present study lend support to the view that BNCT mediated by CuTCPBr and BPA may prove more efficacious than BNCT mediated by either one of those agents.


   Conclusion
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusion
References
 
In mice bearing either of the two murine carcinomas, CuTCPBr appears to be as effective as CuTCPH and considerably more effective than BPA in targeting tumour while maintaining low concentrations in blood and brain tissue. Neither of these porphyrins was toxic to mice at a dose of ~200 mg of porphyrin per kg body weight. At double the dose, ~400 mg porphyrin per kg body weight, there were some mild, transient toxicities discernible up to 1 week after injections of either porphyrin. That CuTCPBr can be labelled by either 64Cu or 76Br for PET imaging suggests that it would be more advantageous than CuTCPH as a tracer in clinical studies given the similar pharmacokinetic and toxicological profiles.


   Acknowledgments
 
We thank Professor J Martin Brown and Dr Mary J Dorie for generously donating the SCCVII cells, Dr Mark W Renner for FT-IR spectra and Dr Kent Riley for prompt-gamma boron analyses at the MITR.


   Footnotes
 
Support for this work was provided by Psimei Pharmaceuticals, Plc., UK and by the US Department of Energy under Contract DE-AC02-76CH00016. Back

Received for publication February 17, 2003. Revision received October 27, 2003. Accepted for publication December 3, 2003.


   References
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusion
 References
 

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