Establishment of a method to determine the magnetic particles in mouse tissues
© Wu et al.; licensee Springer. 2012
Received: 6 October 2012
Accepted: 12 November 2012
Published: 6 December 2012
This work is aimed to evaluate a method to detect the residual magnetic nanoparticles (MNPs) in animal tissues. Ferric ions released from MNPs through acidification with hydrochloric acid can be measured by complexation with potassium thiocyanate. MNPs in saline could be well detected by this chemical colorimetric method, whereas the detected sensitivity decreased significantly when MNPs were mixed with mouse tissue homogenates. In order to check the MNPs in animal tissues accurately, three improvements have been made. Firstly, proteinase K was used to digest the proteins that might bind with iron, and secondly, ferrosoferric oxide (Fe3O4) was collected by a magnetic field which could capture MNPs and leave the bio-iron in the supernatant. Finally, the collected MNPs were carbonized in the muffle furnace at 420°C before acidification to ruin the groups that might bind with ferric ions such as porphyrin. Using this method, MNPs in animal tissues could be well measured while avoiding the disturbance of endogenous iron and iron-binding groups.
KeywordsFerric ions Magnetic nanoparticles Potassium thiocyanate Mouse tissue Chemical colorimetric method
Nanotechnology is widely used in drug or gene delivery and targeted therapy [1–4]. The importance of targeted drug delivery is to transport a drug directly to the center of the disease under various conditions and thereby treat it separately, with less effect on other tissues. The nanoparticle designed for drug delivery should be biodegradable and biocompatible [5, 6]. Due to its good biodegradability and biocompatibility, the engineered magnetic nanoparticles (MNPs) could be well used in disease diagnosis and even in drug delivery and targeted therapy [7–14]. They can be simultaneously functionalized and guided by a magnetic field [15–17]. The safety of designed MNPs depends on the safety of linked molecules and the magnetic cores. So, evaluating how MNPs distribute and metabolize in different tissues of animals is very important. Moreover, this information is capital to give reference of its optimal dosage and administration route. Magnetic resonance imaging, Prussian blue staining, and transmission electron microscopy were used to detect the distribution of the magnetic nanoparticles in vivo. As a vector, MNPs often bond with some conjugate such as cisplatin, and the distribution of conjugates had been used to indicate MNP distribution . Determining iron ions using inductively coupled plasma-mass spectrometry (ICP-MS) is a very sensitive method and has been used to determine the concentration of MNPs in animal tissues . This method could measure the total endogenous and exogenous iron in different tissues of animals. When the tissue contains high concentration of iron ions, the MNP concentration could not be calculated by using this method. Yin et al. have explored the toxicity of Fe3O4 coated with glutamic acid labeled with Fe59 and determined their distribution in mice. Separating from endogenous iron labeled with Fe59 could directly catch the trace of MNPs in the different tissues of mice, including the absorption, distribution and clearance, and accumulation in tissues and the probable target organ, and evaluate its pharmacokinetic profile in vivo. However, with the same disadvantage of ICP mass, labeled Fe59 could not give the information on whether it is a degraded iron ion or an atom in MNPs. Therefore, we try to establish a method to determine the ferric ions in MNPs to observe the metabolism feature of MNPs in animal tissues.
Experimental materials and methods
CD-1 strain mice were supplied by Vital River Laboratory Animal Technology Co. Ltd. (SCXK(Jing)2006-2009, Beijing, China). They were housed in a controlled environment (21 ± 2°C, 55 ± 5% of humidity, 12-h dark/light cycle with light provided between 6 am and 6 pm). Food and water were given ad libitum. All the animal experiments were carried out in the Beijing Center for Drug Safety Evaluation, in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the Center, which is in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International.
The MNPs were from Shanghai Jiaotong Unversity. Iron chloride hexahydrate was from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The MNPs were coated with cetyltrimethyl ammonium bromide at the size of 25 to 35 nm. Magnetic field (MagneSphere Technology Magnetic Separation Stands, Z5341) was from Promega (Madison, WI, USA). Muffle furnace (SX-8-10) was from Tianjin Taisite Instrument Co. Ltd. (Tianjin City, China).
Establishing a method to determine ferric ions in vitro
For quantitive analysis of MNPs the concentration of iron ion was created as a standard for MNPs. Ferric chloride was use to analyze the iron content. Potassium thiocyanide colorimetry showed a good linear relationship and was used to determine ferric ions in MNPs treated with hydrochloride acid at 100°C. Different concentration of 0.5 ml ferric chloride was mixed with an equal volume of 2N hydrochloride acid and boiled for 10 min. The acidified solution was cooled to room temperature and added 120 μl of 5M potassium thiocyanide. Ninety-six-well plates were used in this detection method. The colored reaction product (150 μl/well) was measured at 480 nm on a spectral scanning multimode reader (Varioskan Flash version 2.4.3, Thermo Scientific, Logan, UT, USA). The sonicated and acidified MNPs could be treated in the same way and its ferric ions concentration could be calculated by the ferric chloride standard curve.
The influence of animal tissue on the determination of ferric ions in MNPs
For the assessment of MNPs in different tissues by colorimetry, the normal mice were narcotized with ether. The whole blood was collected into heparin-coated tube and diluted with nine volumes of saline before mixing with MNPs. Different tissues were harvested. Tissues from three mice were mixed and homogenized in ten volumes of ice-cold saline in a homogenizer (ULTRA-TURRAX T25, IKA-Labortechnik, Staufen, Germany) for 10 s. Three hundred microliters of MNPs at 0.078 to 40 μg/ml in saline was mixed with an equal volume of tissue homogenate and acidified with 80 μl of 6N hydrochloric acid. The acidified solution was centrifuged at 14,000 rpm for 6 min, and then, the supernatants were colorized with 80 μl of 5M potassium thiocyanide.
The iron background in mouse tissues
One hundred microliters of 10% mouse tissue homogenate was mixed with 500 μl of saline and then treated with proteinase K at the final concentration of 100 μg/ml under 55°C to 65°C for 0.5 h. The samples were then transferred into crucibles, oven dried on a hot plate, and then carbonized in a muffle at 420°C for 2 h. Cooled to room temperature, the samples were acidified with 0.5 ml 1N hydrochloric acid at 100°C for 10 min. Cooled to room temperature, 1N hydrochloric acid was replenished to the final volume of 1.0 ml. Centrifuged at 10,000 rpm for 10 min, 0.5 ml of the supernatants was colorized with 60 μl of 5M potassium thiocyanide. The concentration of ferric ions was calculated referencing the result from ferric chloride standard.
The determination of MNPs in blood treated with proteinase K with/without magnetic field collection
MNPs at different concentrations in 300 μl of saline were mixed with 30 or 300 μl of mouse whole blood and replenished with saline to the final volume of 600 μl. Aliquots were treated with/without 3 μl of proteinase K, 20 mg/ml, to the final concentration of 100 μg/ml under 55°C to 65°C for 0.5h and centrifuged at 14,000 rpm for 5 min. The 200 μl of the supernatant was mixed with 400 μl of saline to check whether Fe3O4 remained after centrifugation. The precipitate was washed with 0.6 ml saline, centrifuged two times and suspended in 600 μl of saline. Each sample was added with 80 μl 6N hydrochloric acid and boiled for 10 min. After cooling in a water bath at room temperature, the solutions were colorized with 80 μl 5M KSCN, and their A480 were measured.
Using magnetic field to separate MNPs from the endogenous iron followed by carbonation to ruin ferric ions binding groups
Being a standard, 300 μl of MNPs at 40 μg/ml in saline was diluted with 200 μl of saline, acidified with 500 μl 2N hydrochloric acid, and colorized with 120 μl 5M KSCN. The same aliquots of MNPs, 300 μl in saline, were respectively mixed with 200 μl of saline and 200 μl of saline containing 50% mouse blood. The crucibles containing these solutions were put on a magnetic field, and the MNPs were collected and washed two times with saline. The precipitates could be directly acidified with 1 ml 1N hydrochloric acid or carbonized in a muffle furnace at 420°C for 2 h, cooled to room temperature, and then be acidified. After acidification at 100°C for 10 min and cooling to room temperature, all the samples above were replenished and colorized with 120 μl 5M KSCN. Using the same procedure, MNPs mixed in the mouse tissue homogenate was collected and determined. The collected ratio was calculated and compared with the result of the MNPs in saline.
Determination of MNPs in the blood of mice treated with MNPs by intravenous injection
CD-1 mice were i.v.-administrated with MNPs at a dose of 7.5 mg/kg. At different time points after MNP treatment, the whole blood was collected in heparin-coated tube. In a crucible, 0.1 ml of whole blood was mixed with 0.9 ml of saline, and MNPs were collected and washed two times with saline accompanied with magnetic field separation. The collected precipitates were carbonized as before. The residues were suspended in 0.5 ml 1N hydrochloric acid and colorized with 60 μl 5M KSCN. The concentration of ferric ions was calculated, referencing with ferric chloride standard with attention on the volume used.
The standard of ferric ions determination in vitro
The relative standard deviation in the determination of ferric ions (%)
Ferric ions (mg/L)
Within the day
1.90 × 10−3
2.00 × 10−3
The influence of blood content on the measure of MNPs
Ferric ion determination after separating from endogenous iron and ruining ferric ion binding groups
The determination of ferric ions in blood indicated that different treatments affected Fe determination. Even with 5% of whole blood, the A480 decreased significantly, and this decrease could not be alleviated by treating with proteinase K which could digest the protein that might combine with Fe3O4 or Fe3+. These results reminded that there were some organic components in mouse blood that could catch iron, and these components could be released sufficiently after proteinase K treatment. Carbonation is one of the methods that could effectively ruin the groups that might bind with iron from MNPs which were separated from endogenous iron existing in the blood under a magnetic field.
Because nanoparticles (NPs) have the unique physical and chemical properties compared with general substance, their safety could not be well evaluated by normal evaluation methods only. Apart from the toxicity of NPs themselves, the safety of NPs was closely related with the administration way and the distribution path in the animal. Magnetic nanoparticles (MNPs) are drawing increasingly attention due to its unique purposes such as disease diagnosis and drug delivery. Though MNPs could be concentrated at the desired site lead by an external magnet  and have been thought to be biodegradable and biocompatible , the toxicity of iron in animals should not be ignored because overload of iron oxide has potential toxicity to blood, liver, spleen and kidneys [19, 23], especially when the iron was in nanoscale. The limited studies on MNPs toxicity were pointed at the influence of MNP construction, size, surface chemistry and the design of their administration in the toxic outcomes as anticipated . Establishing an experimental method to detect the concentration of MNPs in different tissue and organ of animals is very important to obtain the rule of MNPs distribution and metabolism and then to suspect the potential toxicity.
In this study, chemical colorimetric method to determine the residual MNPs in the tissues of mice has been established. Whole blood was selected as the tissue to establish the MNPs determination method because of the blood half-life is a capital value indicating whether MNPs could evade uptake by RES [25–28]. Compared with other tissues, MNPs in blood was relatively difficult to be measured because the red blood cells contain large amounts of iron. Some research reported that the MNPs in the plasma could be separated with blood cell by centrifugation . It is possible that the MNPs could aggregate and co-precipitate with blood cells. In this research the iron backgrounds in different tissues of mouse were determined elucidating the high bio-iron background in whole blood, spleen, liver and even in intestine. Using ICP mass MNPs, signals in these samples could not be detected accurately especially when lower dose of MNPs was used. So using a magnet to separate MNPs from bio-iron is very important. Ultrasound was used to avoid the aggregation of MNPs and to break the blood cells preventing their precipitation on magnetic field. Proteinase K is effective enzyme to digest proteins might bind with MNPs. Carbonation in muffle and then acidification could oxidize ferrous ions to ferric ions which would be colorized by KSCN. The groups that might complex with ferric ions in tissues could be destroyed when the sample was carbonated. This method will be used to measure the MNPs distribution and metabolism in mice in our further research, especially to validate whether this method could be used to check the MNPs coupled with antibody or other medicine groups.
In this study, we established a method to determine MNPs in mouse tissues without the disturbance of endogenous iron. Even being little tedious, the method is safe and effective. Following more and more uses of MNPs in vivo take place, this method could be of great assistance in measurements of MNPs itself and MNPs coupled with drug or antibody, especially when the tissue containing high level iron and the animal treated with lower dose of MNPs.
YW, a senior student at the School of Medicine, Shanghai Jiaotong University, voluntarily worked as a research assistant in the Beijing Institute of Pharmacology and Toxicology during the summer break. YW, is a professor engaged in biochemical pharmacology. JW is a technician engaged in immunological analysis and works with YW. WZ, QL, ND, and ZG were graduate students in the laboratory of YW in the Beijing Institute of Pharmacology and Toxicology. GG, HH, and KW are doing research with DC in the Department of Bio-Nano Science and Engineering, Shanghai Jiaotong University.
This work was supported by The National Basic Research Program of China (no. 2010CB933904) and grants from the National Natural Science Foundation of China (no. 30973562).
- Whitesides GM: The ‘right’ size in nanobiotechnology. Nat Biotechnol 2003, 21: 1161–1165. 10.1038/nbt872View ArticleGoogle Scholar
- Sengupta S, Eavarone D, Capila I, Zhao GL, Watson N, Kiziltepe T, Sasisekharan R: Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system. Nature 2005, 436: 568–572. 10.1038/nature03794View ArticleGoogle Scholar
- Pack DW, Hoffman AS, Pun S, Stayton PS: Design and development of polymers for gene delivery. Nat Rev Drug Discov 2005, 4: 581–593. 10.1038/nrd1775View ArticleGoogle Scholar
- Cai D, Mataraza JM, Qin ZH, Huang ZP, Huang JY, Chiles TC, Carnahan D, Kempa K, Ren ZF: Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing. Nat Methods 2005, 2: 449–454. 10.1038/nmeth761View ArticleGoogle Scholar
- Gupta AK, Gupta M: Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26: 3995–4021. 10.1016/j.biomaterials.2004.10.012View ArticleGoogle Scholar
- Xie J, Xu C, Xu Z, Hou Y, Young KL, Wang SX, Pourmand N, Sun S: Linking hydrophilic macromolecules to monodisperse magnetite (Fe3O4) nanoparticles via trichloro-s-triazine. Chem Mater 2006, 18: 5401–5403. 10.1021/cm061793cView ArticleGoogle Scholar
- Mishra B, Patel BB, Tiwari S: Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery. Nanomedicine 2010, 6: 9–24. 10.1016/j.nano.2009.04.008View ArticleGoogle Scholar
- Suh WH, Suslick KS, Stucky GD, Suh YH: Nanotechnology, nanotoxicology, and neuroscience. Prog Neurobiol 2009, 87: 133–170. 10.1016/j.pneurobio.2008.09.009View ArticleGoogle Scholar
- Stepp P, Thomas F, Lockman PR, Chen H, Rosengart AJ: In vivo interactions of magnetic nanoparticles with the blood–brain barrier. J Magn Magn Mater 2009, 321: 1591–1593. 10.1016/j.jmmm.2009.02.092View ArticleGoogle Scholar
- Mailander V, Landfester K: Interaction of nanoparticles with cells. Biomacromolecules 2009, 10: 2379–2400. 10.1021/bm900266rView ArticleGoogle Scholar
- Kral V, Sotola J, Neuwirth P, Kejik Z, Zaruba K, Martasek P: Nanomedicine—current status and perspectives: a big potential or just a catchword? Chem List 2006, 100: 4–9.Google Scholar
- Zhang XQ, Jiang L, Zhang CL, Li D, Wang C, Gao F, Cui DX: A silicon dioxide modified magnetic nanoparticles-labeled lateral flow strips for HBs antigen. J Biomed Nanotechnol 2011, 7: 776–781. 10.1166/jbn.2011.1352View ArticleGoogle Scholar
- Li S, Liu HN, Jia YY, Deng Y, Zhang LM, Lu ZX, He N: A novel SNPs detection method based on magnetic nanoparticles array and single base extension. Theranostics 2012, 2: 967–975. 10.7150/thno.5032View ArticleGoogle Scholar
- Liu H, Li S, Liu LS, Tian L, He NY: An integrated and sensitive detection platform for biosensing application based on Fe@Au magnetic nanoparticles as bead array carries. Biosens Bioelectron 2010, 26: 1442–1448. 10.1016/j.bios.2010.07.078View ArticleGoogle Scholar
- Gui C, Dai X, Cui DX: Advances of nanotechnology applied to biosensors. Nano Biomed Eng 2011, 3: 260–273.View ArticleGoogle Scholar
- Cui DX, Han YD, Li ZM, Song H, Wang K, He R, Liu B, Liu HL, Bao CC, Huang P, Ruan J, Gao F, Yang H, Cho HS, Ren QS, Shi DL: Fluorescent magnetic nanoprobes for in vivo targeted imaging and hyperthermia therapy of prostate cancer. Nano Biomed Eng 2009, 1: 61–74.View ArticleGoogle Scholar
- Ch M, Li CY, He NY, Wang F, Ma NN, Zhang LM, ZhX L, Ali Z, ZhJ X, Li XL, Liang GF, Liu HN, Deng Y, Xu LJ, ZhF W: Preparation and characterization of monodisperse core-shell Fe3O4@SiO2 microspheres and its application for magnetic separation of nucleic acids from E. coli BL21. J Biomed Nanotechnol 2012, 8: 1000–1005. 10.1166/jbn.2012.1454View ArticleGoogle Scholar
- Zhang HZ, Xie MQ, Kang Z, Shen H, Wang L: Targeted distribution of cis-platin magnetic nanoparticles in mice. J South Med Univ 2008, 28: 1756–1763.Google Scholar
- Jain TK, Reddy MK, Morales MA, Leslie-Pelecky DL, Labhasetwar V: Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats. Mol Pharm 2008, 5: 316–327. 10.1021/mp7001285View ArticleGoogle Scholar
- Yin QH, Liu L, Gu N, Huang Y, Liu L, Song JH, Cui Y: Determining the biodistribution of nano-59Fe-Fe2O3-Glu in mice by 59Fe tracer and preparation. J Medical Postgraduate 2005, 18: 312–317.Google Scholar
- Kresse M, Wagner S, Pfefferer D, Lawaczeck R, Elste V, Semmler W: Targeting of ultrasmall superparamagnetic iron oxide (USPIO) particles to tumor cells in vivo by using transferrin receptor pathways. Magn Reson Med 1998, 40: 236–242. 10.1002/mrm.1910400209View ArticleGoogle Scholar
- Wen M, Song L, Wei B, Li SL, Li BB: Preparation of superparamagnetic iron oxide nanoparticles and its acute toxicity to mice. Acad J Sec Mil Med Univ 2007, 28: 1104–1108.Google Scholar
- Zhai Y, Wang XL, Wang XM, Xie H, Gu HC: Acute toxicity and irritation of water-based dextran-coated magnetic fluid injected in mice. J Biomed Mater Res A 2008, 85: 582–587.Google Scholar
- Nel A, Xia T, Mädler L, Li N: Toxic potential of materials at the nanolevel. Science 2006, 311: 622–627. 10.1126/science.1114397View ArticleGoogle Scholar
- Chen LT, Weiss L: The role of the sinus wall in the passage of erythrocytes through the spleen. Blood 1973, 41: 529–537.Google Scholar
- Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, Bawendi MG, Frangioni JV: Renal clearance of quantum dots. Nat Biotechnol 2007, 25: 1165–1170. 10.1038/nbt1340View ArticleGoogle Scholar
- Decuzzi P, Ferrari M: The adhesive strength of non-spherical particles mediated by specific interactions. Biomaterials 2006, 27: 5307–5314. 10.1016/j.biomaterials.2006.05.024View ArticleGoogle Scholar
- Storm G, Belliot SO, Daemen T, Lasic DD: Surface modification of nanoparticles to oppose uptake by the mononuclear phagocyte system. Adv Drug Deliv Rev 1995, 17: 31–48. 10.1016/0169-409X(95)00039-AView ArticleGoogle Scholar
- Gao WH, Liu ST, Fan CX, Qi LY, Chen ZL: Pharmacokinetics, tissue distribution and magnetic resonance's response characteristics of folic acid-O-carboxymethyl chitosan ultrasmall superparamagnetic iron oxide nanoparticles in mice and rats. Acta Pharmaceutica Sinica 2011, 46: 845–851.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.