|Year : 2012 | Volume
| Issue : 2 | Page : 204-208
Evaluation of radiogallium-labeled, folate-embedded superparamagnetic nanoparticles in fibrosarcoma-bearing mice
Seyyedeh Leila Hosseini-Salekdeh1, Amir Reza Jalilian2, Hassan Yousefnia2, Kammaledin Shafaii3, Majid Pouladian1, Morteza Mahmoudi4
1 Department of Engineering and Technical, Science and Research Branch, Islamic Azad University, Tehran, 14778-93855, Iran
2 Radiopharmaceutical Research and Development Lab (RRDL), Nuclear Science and Technology Research Institute (NSTRI), Tehran, 14155-1339, Iran
3 Agricultural, Medical and Industrial Research School (AMIRS), Nuclear Science and Technology Research Institute (NSTRI), Tehran, Karaj, Postal code: 31485-498, Iran
4 Department of Materials Science and Engineering, Sharif University of technology, Tehran, 11365-8639, Iran
|Date of Web Publication||26-Jul-2012|
Radiopharmaceutical Research and Development Lab (RRDL), Nuclear Science and Technology Research Institute (NSTRI), Tehran, 14155-1339
Source of Support: Nuclear Science and Technology Research Institute (NSTRI), Conflict of Interest: None
Context: Elevated expression of the folate receptor (FR) occurs in many human malignancies. Thus, folate targeting is widely utilized in drug delivery purposes specially using nano-radioactive agents.
Aims: In this work, we report production and biological evaluation of gallium-67 labeled superparamagnetic iron oxide nanoparticles, embedded by folic acid ( 67 Ga-SPION-folate) complex especially in tumor-bearing mice for tumor imaging studies.
Settings and Design: The structure of SPION-folate was confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM) and foureir transform infrared spectroscopy (FT-IR) analyses. The radiolabeled SPION-folate formation was confirmed by instant thin layer chromatography (ITLC). Tumor induction was performed by the use of poly-aromatic hydrocarbon injection in rodents as reported previously.
Materials and Methods: [ 67 Ga]-SPION-folate was shown to possess a particle size of ≈5-10 nm using instrumental methods followed by ITLC test. Biocompatibility of the compound was investigated using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay followed by stability tests and tumor accumulation studies in fibrosarcoma-bearing mice after subcutaneous (s.c.) application.
Statistical Analysis Used: All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student t-test. Statistical significance was defined as P<0.05.
Results: [ 67 Ga]-SPION-folate was prepared by a modified co-precipitation method possessing a particle size of ≈5-10 nm using instrumental methods (>95% radiochemical purity). Biodistribution studies demonstrated tumor:blood, tumor:bone and tumor:muscle ratios of 4.23, 4.98 and 11.54 respectively after 24 h.
Conclusions: Due to the nano-scale size and high-penetrative property of the developed folate-containing nano-complex, this system can be an interesting drug delivery modality with therapeutic applications and folate receptor-targeting behavior, while possessing paramagnetic properties for thermotherapy.
Keywords: Biodistribution, fibrosarcoma, folate, Ga-67, SPION
|How to cite this article:|
Hosseini-Salekdeh SL, Jalilian AR, Yousefnia H, Shafaii K, Pouladian M, Mahmoudi M. Evaluation of radiogallium-labeled, folate-embedded superparamagnetic nanoparticles in fibrosarcoma-bearing mice. J Can Res Ther 2012;8:204-8
|How to cite this URL:|
Hosseini-Salekdeh SL, Jalilian AR, Yousefnia H, Shafaii K, Pouladian M, Mahmoudi M. Evaluation of radiogallium-labeled, folate-embedded superparamagnetic nanoparticles in fibrosarcoma-bearing mice. J Can Res Ther [serial online] 2012 [cited 2020 Mar 31];8:204-8. Available from: http://www.cancerjournal.net/text.asp?2012/8/2/204/98971
| > Introduction|| |
Folate is necessary for the production and maintenance of new cells.  Elevated expression of the folate receptor (FR) occurs in many human malignancies, especially when associated with aggressively growing cancers,  such as ovarian tumors,  and other gynecological cancers. , The application of the various forms of iron oxides (i.e. magnetite and maghemite) for radiological diagnostic procedures, to document vascular leakage, macrophage imaging or cell tracking has been reported. Also, the small size of nanomaterials allows better surface functions as well as cell permeability.  In this work, we report biological evaluation of [ 67 Ga]-SPION-folate complex especially in tumor-bearing mice.
| > Materials and Methods|| |
[ 67 Ga] was supplied from Agricultural, Medical and Industrial Research School (AMIRS) 30 MeV cyclotron (Cyclone-30, IBA). ITLC was performed by Whatman No. 1. (Maidstone, U.K.), using a thin layer chromatography scanner, Bioscan AR2000, Paris, France. Calculations were based on the 184 keV peak for [ 67 Ga]. All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student t-test. Statistical significance was defined as P<0.05. Animal studies were carried out in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, 2nd ed. (approved by Iranian Ministry of Health and Medical Education).
In this study, we have used a different method to produce SPION-folate compared to previous studies. , Solutions were prepared using deionized (DI) water after 30 min bubbling under an N 2 atmosphere. The iron salts were dissolved in DI water containing 0.5 M HCl where the mole fraction of Fe 2+ to Fe 3+ was adjusted to 1:2 (1 g of FeCl 3 :0.368 g of FeCl 2 ) for all samples. The precipitation was performed by drop wise addition of iron salt solutions to NaOH solutions under an N 2 atmosphere. In order to control mass transfer, which may allow particles to combine and build larger polycrystalline particles, turbulent flow was created by placing the reaction flask in an ultrasonic bath and changing the homogenization rates between 3600 and 9000 rpm in the first 2 min of the reaction. The molarities of the NaOH solution and the stirring rate were fixed at 2.4 and 9000 rpm, respectively. The details of experiments applied for choosing levels of these parameters have been reported elsewhere. After 30 min, a solution of folic acid (1 mg/ml, 100 μl) with a pH of 8 and citrate buffer were added by a syringe, and the reaction mixture was stirred at 3600 rpm for an additional 30 min. The mixture was then sonicated for 20 min in an ultrasound bath. The particles were collected by centrifugation at 6000 rpm for 10 min and redispersed in DI water (several times). The filtered mass was dried in oven for 72 h at 40°C. A fraction of SPION-folate was dried to obtain a membranous solid sample and then the solid sample was used for XRD, VSM and FT-IR analyses. The final sample structure was determined using TEM (ZEISS Model EM-10C) operating at 100 kV, high resolution scanning electron microscope (HRSEM) (FEG LECO) for size and morphology characterization. XRD (Siemens D5000) with Cu Kα radiation was used for the phase characterization and particle size determination.  FTIR spectra of the samples were taken in KBr pellets using an ABB Bomem MB-100 FTIR spectrophotometer. The magnetization of the samples in a variable magnetic field was measured using a vibrating sample magnetometer (VSM) with a sensitivity of 10 -3 emu and magnetic field up to 8 kOe. The magnetic field was changed uniformly with a time rate of 66 Oe/s.
In vitro biocompatibility assessment test using MTT assay was performed according to the previously reported method. 
Radiolabeled SPION-folate was prepared by using the procedure mentioned previously with modifications. Briefly, the solution of [ 67 Ga] chloride (1 mCi of [ 67 Ga]-chloride in 0.2M HCl) was evaporated using N 2 gas followed by adding prepared iron salts solution to the [ 67 Ga] vial. The precipitation was performed by sodium hydroxide under the conditions mentioned above and followed by neutralization. The final pH of the solution was adjusted to 6.8 using acetate buffer and the mixture was passed through a 0.22 μ filter. The solution was then used for biological studies. The radiochemical purity of the nanoparticles was determined using Whatman chromatography paper (Whatman No. 1. Whatman, Maidstone, UK), and developed in a mixture of methanol/water/acetic acid (4:4:2) as the mobile phase.
Tumor induction was performed by the use of poly-aromatic hydrocarbon injection in rodents as reported previously.  For tumor model preparation, 10 μl of 3-methyl cholanthrene solution in extra-virgin olive oil (4 mg/ml) was injected s.c to the dorsal area of the mice. After 14-16 weeks, the tumor weighed 0.2-0.4 g and was not grossly necrotic. Tumor tissues of some random animals were sent for pathological tests and were diagnosed as fibrosarcoma.
To determine its biodistribution, [ 67 Ga]-SPION was administered s.c. to fibrosarcoma-bearing mice. A volume (50-100 μl) of final [ 67 Ga]-SPION solution containing 20 μCi radioactivity was injected subcutaneously under mice tail skin. The animals (n=3) were sacrificed at exact time intervals (4, 24 and 48 h), and the specific activity of different organs was calculated as percentage of tissue count per gram using an HPGe detector.
| > Results|| |
Like SPION-folate synthesis and analysis done in previous work,  these processes were reported again including TEM, HRSEM, FT-IR, XRD and VSM tests.
[Figure 1] shows TEM images of SPION-folate synthesized. As seen, the nanoparticles with narrow size distribution have been achieved. The average particle sizes from the TEM micrographs were determined to be about 5-10 nm.
[Figure 2] shows HRSEM images of SPION-folate synthesized. As seen, the nanoparticles with narrow size distribution have been achieved. The average particle sizes from the HRSEM micrographs were determined to be about 5-10 nm.
The result of FT-IR has been reported previously.  SPION-folate IR spectrum demonstrated shifted peaks for various functional groups for both starting materials. For instance, folate amide and carboxylic acid groups peaks were all shifted to smaller wave numbers (1447cm -1 ) compared with folic acid (data not shown). In SPION IR spectrum, a broad peak at 565 cm -1 was observed which was shifted to 607 for SPION-folate [Figure 3].
[Figure 4] shows XRD patterns of the synthesized nanoparticles. The particles showed significant peak broadening and quasi-crystalline pattern due to the formation of extremely small particles, i.e. 5 nm (few atomic layers). Defective structure of the SPION-folate may also diffuse the X-ray reflections; in addition, no change in the XRD patterns was observed by repeating the synthesized method for three times. Scherrer method has been used to determine the particle size and the result was 5 nm.
|Figure 4: XRD patterns of SPION-folate showing the formation of magnetite|
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[Figure 5] shows the hysteresis loop of SPION-folate particles determined by VSM. The particles exhibit superparamagetic behaviors, i.e. a negligible remanence and coercivity. The superparamagnetic behavior has a crucial case concerning SPION compounds in order to use them in biomedical applications especially in hyperthermia.
|Figure 5: Magnetization curve for SPION-folate showing superparamagnetic behavior for both species|
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The experiments to confirm SPION-folate biocompatibility were performed according to the literature.  Interestingly, incorporation of the folate moiety in the SPION structure increased the cell viability and also possibly the biocompatibility of the particles [Figure 6].
|Figure 6: Cell viability of bare-SPION and SPION-folate at administered concentrations 3-48 h|
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Many methods were employed to obtain the radiolabeled-SPION-folate and the only successful way with the lowest possible alteration showed to be the incorporation of the radionuclide within the nanostructure of iron oxide network under the synthesis procedure. Since free amounts of radioisotopes were used in carrier, there must have been no significant changes in the nanoparticle structure of interest.
The radiolabeled SPION-folate has higher binding with polar silicate solid phase while keeping Ga-67 in their inner structure and remaining at the lower R f (R f . 0.3) as shown in [Figure 7], while free 67 Ga 3+ moves to higher R f (R f 0.8).
|Figure 7: RTLC chromatogram of a 67Ga-SPION-folate sample on Whatman paper|
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The distributions of [ 67 Ga]GaCl 3 , [ 67 Ga]-SPION and [ 67 Ga]-SPION-folate among wild type rat tissues have already been reported.  For biodistribution among tumor-bearing mice, 0.1 ml of final solution containing 20 μCi radioactivity was injected under the tail skin. The total amount of radioactivity injected into each mouse was measured by counting the 1-ml syringe before and after injection in a dose calibrator with a fixed geometry. The animals were sacrificed by CO 2 asphyxiation at selected times after administration of anesthesia using propofol: ketamine mixture, the tissues were weighed and their specific activities were determined with a -ray scintillation as a percent of count per gram of tissue. Although the data were collected from 2 to 48 h post injection (data not shown), the most valid data were obtained after 24 h [Figure 8].
|Figure 8: Biodistribution of [67Ga]-SPION-folate in fi brosarcoma-bearing mice 24 h after subcutaneous injection (n=3)|
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| > Discussion|| |
By taking into account the structural characterization and magnetic measurements, it was shown that SPION-folate particles in this study were well prepared. An MTT assay was used to investigate the biocompatibility of folate-coated SPION using L929 cells. Total labeling and formulation of [ 67 Ga]-SPION-folate took about 60 min, with a radiochemical purity >95%. The final preparation was administered to fibrosarcoma-bearing mice subcutaneously and the biodistribution of the radiotracer was checked up to 48 h post-injection. The tumor:blood, tumor:bone and tumor:muscle ratios were 4.23, 4.98 and 11.54 respectively after 24 h post s.c. injection. In our previous work,  we showed that the SPIONs in combination with folate could have better bioavailability than bare
SPIONs.  On the other hand, since this investigation is carried out by VSM and magnetization property changes from -60 to 60 (emu/g) [Figure 5], similar to previous works, , these particles are paramagnetic and paramagnetic property is not altered by coating process.
Cell viabilities of coated SPION were found to be higher than the bare SPION. This happens due to (a) good biocompatibility of the coating compound and (b) much lower and positive electric charges on the coated particles, in contrast with negative charges for a bare one, which cause limited available sites for other compounds in cell culture medium such as Cl− ions (zeta potential was −21.14 and 3.24 for bare and coated SPION, respectively). 
Due to the nano-scale size and high-penetrative property of the developed folate-containing nano-complex, this system can be an interesting drug delivery modality with therapeutic applications and folate receptor-targeting property, while possessing paramagnetic properties for thermotherapy. Application of hydrophilic polymers as coating agents on SPIONs can reduce liver uptake.
| > References|| |
|1.||Kamen B. Folate and antifolate pharmacology. Semin Oncol 1997;24 (5 Suppl 18):S18-30-S18-39. |
|2.||Campbell IG, Jones TA, Foulkes WD, Trowsdale J. Folate-binding protein is a marker for ovarian cancer. Cancer Res 1991;51:5329-38. |
|3.||Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 2005;338:284-93. |
|4.||Wu M, Gunning W, Ratnam M. Expression of folate receptor type A in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. J Cancer Epidemiol Biomark Prev 1999;8:775-83. |
|5.||Dainty LA, Risinger JI, Morrison C. Overexpression of folate binding protein and mesothelin are associated with uterine serous carcinoma. J Gynecol Oncol 2007;105:563-70. |
|6.||Mahmoudi M, Simchi A, Milani AS, Stroeve P. Cell toxicity of superparamagnetic iron oxide nanoparticles. J Colloid Interface Sci 2009;336:510-8. |
|7.||Mahmoudi M, Simchi A, Imani M. Cytotoxicity of Uncoated and Polyvinyl Alcohol Coated Superparamagnetic Iron Oxide Nanoparticles. J Phys Chem 2009;113:9573-80. |
|8.||Jalilian AR, Hosseini-Salekdeh SL, Mahmoudi M, Yousefnia H, Majdabadi A. Preparation and Biological Evaluation of radiolabeled-folate embedded Superparamagnetic Nanoparticles in wild-type Rats. J Radioanal Nucl Chem 2011;287:119-27. |
|9.||Fowlkes WY, Creveling CM. Engineering methods for robust product design: Using taguchi methods in technology and product development. New York: Prentice Hall; 1995. |
|10.||Jalilian AR, Panahifar A, Mahmoudi M, Akhlaghi M, Simchi A. Preparation and Biological Evaluation of [ 67 Ga]-labeled-Superparamagnetic Nanoparticles in normal Rats. J Radiochim Acta 2009;97:51-6. |
|11.||Nowostawska M, Corr SA, Byrne SJ, Conroy J, Volkov Y, Gun'ko YK. Porphyrin-magnetite nanoconjugates for biological imaging. J Nanobiotechnology 2011;9:13-24. |
|12.||Mahmoudi M, Simchi A, Imani M, Milani AS, Stroeve P. An in vitro study of bare and poly(ethylene glycol)-co-fumarate-coated superparamagnetic iron oxide nanoparticles: A new toxicity identification procedure. Nanotechnology 2009;20:225104. |
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