- Email: [email protected]
Cytotoxicity of selected magnetic fluids on human adenocarcinoma cells
Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Cytotoxicity of selected magnetic fluids on human adenocarcinoma cells Ingrid Hilgera,*, Sylvia Fru. haufa, Werner Linb, Robert Hiergeistc, Wilfried Andr.ac, Rudolf Hergtc, Werner A. Kaisera a
Institut fu. r Diagnostische und Interventionelle Radiologie des Klinikums der Friederich-Schiller-Universita.t Jena, Bachstraße 18, D-07740 Jena, Germany b Institut fu.r Anatomie I der Friederich-Schiller-Universita.t Jena, Teichgraben 7, D-07740 Jena, Germany c Institut fu.r Physikalische Hochtechnologie Jena, Winzerlaer Straße 10, D-07740 Jena, Germany Received 3 January 2001; received in revised form 30 March 2001
Abstract Based on the knowledge that the magnetite particles seem to be well tolerated by the human body, the cytotoxic potential of coated particles was investigated, which had been selected for potential applications regarding the minimalinvasive elimination of breast tumors by magnetic thermoablation. Human adenocarcinoma cells (BT-20) were exposed (24, 48 and 72 h) to different magnetite particles with diverging total size (8, 10 and 220 nm) and coating (cationic and anionic). One sample contained only non-coated magnetite particles. The magnetite concentration ranged between 0.2 and 20 ng/cell. Cytotoxicity was estimated by measuring the succinate dehydrogenase activity. The morphologic features resulting from the interaction of magnetic fluids with BT-20 cells was determined by transmission electron microscopy. As opposed to the non-coated magnetic particles, cationic particles induced the strongest decrease in cell survival rates depending on time and concentration. Morphologically, the cationic particle samples exerted a strong binding to cellular membranes. Changes in the subcellular structure were found in relation to the coated magnetic particles. In conclusion, our results show that the coated prototype magnetic particles, particularly those with a cationic surfactant, are cytotoxic to BT-20 cells. The cytotoxicity is attributed to electrostatic bindings with cellular membranes, influences of chemical components or non-physiologic pH. Considering the in vivo applications, adverse systemic effects are conceivable and more biocompatible coatings for the selected magnetic particles should be elaborated. r 2001 Elsevier Science B.V. All rights reserved. Keywords: Magnetic fluids; Cytotoxicity; Magnetic thermoablation; Breast cancer; Cancer therapy; Heating
1. Introduction Since the past decade, there has been an increasing interest in the use of colloidal suspen*Corresponding author. Tel.: +49-3641-935037; fax: +493641-936767. E-mail address: [email protected] (I. Hilger).
sions of magnetic nanoparticles for quite different purposes, e.g. cell separation techniques [1], biochemical analysis [2], diagnostic [3] or therapeutic applications [4]. Considering the previous investigations (e.g. Ref. [5]), a procedure called magnetic thermoablation, is being elaborated that possibly allows the minimal-invasive elimination of breast tumors. The fundamentals of the method
0304-8853/03/$ - see front matter r 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 1 ) 0 0 2 5 8 - X
8
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Table 1 Characteristics of magnetic particles used in the present study. The specific absorption rate (SAR) is defined by the energy converted into heat by magnetic losses per time and magnetite mass and was measured for an alternating magnetic field of 400 kHz and 6.5 kA/m Sample
Average total particle size (nm)
SAR (W/g)
Components of coating
1 2 3 4
10 10 220 8
82 69 29 38
Organic components with negative surfactant Organic components with positive surfactant Starch derivative with positive (DEAE) surfactant None
are that after loading the tumor with a magnetic material (iron oxides, magnetite) and exposing the breast to an alternating magnetic field, heating is generated in order to kill the tumor cells. The basic feasibility of the technique has been studied in previous investigations [6,7]. An important prerequisite for applications in magnetic thermoablation is the heating capabilities of the magnetite samples to be used which were shown to vary considerably between each other [8]. The heating capabilities are described by the specific absorption rate (SAR) being the energy converted into heat by magnetic losses per time and mass. Using the selected prototype magnetic particles with adequate heating potentials, temperatures of up to 701C were recorded in adenocarcinomas implanted in mice [9]. Nevertheless, up to now it is not known if the prototype samples are biocompatible to living organisms. Whereas magnetite per se is known to be well tolerated by the human body (e.g. Ref. [10]), magnetite nanoparticles are mostly maintained in colloidal suspensions using particle coatings with largely unspecified biocompatibility features. Therefore, in the present investigation, we investigated the cytotoxic potency after incubation of human adenocarcinoma cells with different prototype colloidal solutions of magnetic nanoparticles which, according to previous investigations, are of interest for applications regarding magnetic thermoablation of breast tumors. 2. Materials and methods 2.1. Magnetic nanoparticle samples Four different water-based prototype magnetic nanoparticle suspensions (ferrofluids) were used
(samples 1, 2 and 4FFerrofluidics: Nu. rtingen, Germany; sample 3 was kindly provided by Chemicell, Berlin, Germany). The samples differed from each other by both the average total particle size and particle coating. The reference sample (sample 4) was composed of non-coated magnetite particles. The SAR of different magnetite samples was determined from time dependent calorimetric measurements in an alternating magnetic field (amplitude of 14 kA/m and 300 kHz frequency) [8]. The particle characteristics are summarized in Table 1. 2.2. Cell culturing An asynchronically growing human breast adenocarcinoma cell line (BT-20, Deutsches Krebsforschungszentrum: Heidelberg, Germany) was cultivated in an exponential monolayer growth. The cultures were maintained in MEM containing 10% (v/v) fetal calf serum (Life Technologies: Karlsruhe, Germany) and 1 mM sodium pyruvate (GibcoBRL: Karlsruhe, Germany). All cultures were routinely checked for mycoplasma contamination. Cells were used up to the 30th passage. 2.3. Determination of cell viability after exposure of BT-20 cells to magnetic nanoparticles Cells were seeded in 96 microtiter plates (density: 1 103 cells/well). After 24 h, the culture medium was replaced by the magnetic particles containing one. The magnetite concentration was adjusted to be 0.2, 2 and 20 ng/cell (corresponding to the concentrations between 2 and 200 mg/ml culture medium). After 24, 48 and 72 h of incubation, the cytotoxicity of the samples was
9
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
determined using the succinate dehydrogenase assay [11]. Survival rates (v) were calculated by the formula: v(%)=(t 100%) : k, where t means the absorbance (450 nm) of the exposed cells and k of the non-exposed control cells.
2.4. Determination of morphologic features after the exposure of BT-20 cells to magnetic nanoparticle samples 1 106 cells were seeded in culture flasks (25 cm2) and incubated under standard conditions. After 24 h, cells were washed 3 times with PBS, incubated with the different magnetic nanoparticle samples (magnetite concentration: 30 ng/cell, equivalent to 6 mg/ml culture medium) for 4 min under room temperature. After washing 4 times with PBS in order to remove unbound magnetic particles, cells were fixed with a 3% (v/v) glutaraldehyde (Agar Scientific LTD: Stansted/ Essex GB) solution and processed for electron microscopy examinations.
3. Results Different effects of the analyzed prototype ferrofluid samples on the cell survival rates were observed (Table 2). Sample 4 showed only a slight decrease of the cell survival rates at higher magnetite mass densities (e.g. 20 ng/cell). These effects were more pronounced with increasing incubation time. Similar relationships were observed for sample 1. Samples 2 and 3 having a cationic particle surfactant revealed both distinct time as well as magnetite-concentration dependent effects on the viability of cells. In particular, no viable cells were found after the application of 20 ng/cell of sample 2. Therefore, sample 2 seems to be more cytotoxic to cells than sample 4. Particularly, comparing the cell survival rates for a concentration of 20 ng/cell, the cell survival rates related to samples 2 and 3 were lower than those for samples 1 and 4. The electron microscopy examinations (Fig. 1) of cells incubated for 4 min at a concentration of 30 ng/cell showed a continuous congregation of
Table 2 Cell survival of BT-20 cells after incubation with different magnetite nanoparticle samples for 24, 48 and 72 h. Experiments were performed in triplicate Sample/magnetite concentration (ng/cell)
Cell survival rates after incubation times (in h) 24
48
72
Sample 1 0.2 2 20
9276 9674 8974
8573 8577 7974
8577 8878 7977
Sample 2 0.2 2 20
9775 375 070
8478 372 070
8875 070 070
Sample 3 0.2 2 20
10575 9374 6976
99711 9078 5975
9174 7674 3672
Sample 4 0.2 2 20
9974 9275 9275
10377 9577 9174
9775 9876 8277
10
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Fig. 1. Subcellular features after exposure of BT-20 cells to different magnetite nanoparticle samples. (1)–(4): results with regard to samples 1–4 (30 ng magnetite/cell for 4 min), respectively. In contrast to (4), cells depicted in (1)–(3) show altered cellular features. ArrowsFcontinuous concentration of mostly aggregated and electron-dense particles along the cell membranes; arrowheadsFirregularly distributed agglomerations of electron-dense particles at the plasmalemma; vFvacuoles; iFmembrane-delimited, electrondense bodies (presumably lysosomes); cFchromatin, ‘‘clotted’’F(2) and (3) or normalF(4); nFnucleoli, ‘‘clotted’’F(2) and (3), normalF(4); oFmitochondria, swollenF(3), normalF(4); eFvacuolar structures of the endoplasmic reticulum; dFdiscontinuous plasmalemma. Magnification: approximately 5000-fold (technically caused variations among 10%).
mostly clustered and electron-dense particles along the cell membranes exposed to cationic magnetic particles (samples 2 and 3) containing culture medium. In contrast to that, irregularly distributed agglomerations of electron-dense particles at the plasmalemma were found in cells exposed to (samples 4 and 1). The subcellular features after incubation with sample 1 revealed a homogeneous distribution of chromatin within the cell nuclei and vacuolar alterations of the endoplasmic reticulum together with rounded electron-dense inclusions in the cytoplasm. The mitochondria were found to be partially swollen. With regard to sample 2, subcellular alterations typical for dying cells were found, such as discontinuously distributed chromatin in the cell nuclei, predominating vacuoles in the cytoplasm and disrupted cell membranes. The subcellular alterations found in relation to sample 3 included the presence of mostly swollen mitochondria, widespread vacuolar structures, welldelimited vesicles and tubes of the endoplasmic
reticulum, membrane-delimited and electron-dense bodies (presumably lysosomes) in the cytoplasm. The chromatin was in parts homogeneously disseminated within the cell nuclei, and partially distributed like clods. With regard to sample 4, no cellular alterations were found: cell nuclei and cytoplasm were inconspicuous and the mitochondria intact.
4. Discussion The cytotoxic potency of the different prototype ferrofluids was assessed by the determination of the cell survival rates as well as by the morphologic features resulting from the interaction with cells. According to our data, the strongest cytotoxic potency, illustrated by time and magnetite-concentration dependent decreases of cell survival, was found for samples distinctively aggregating to cell membranes, as was observed with regard to the cationic magnetic material (samples 2 and 3).
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
The irregularly distributed particle accumulations in the proximity of cell membranes found in relation to samples 1 and 4 are mainly attributed to the preparation of artifacts during cell sample processing for electron microscopy examinations. According to Shinkai et al. [12], growth inhibition was not observed after incubation of a rat glioma cell line with 200 ng of magnetite cationic liposomes per cell, but at higher magnetite cell concentrations. The exposure time was up to 80 h. In comparison to that, our results indicate that samples 2 and 3 are of higher cytotoxicity than the aforementioned magnetite cationic liposomes. The components of the coating being biocompatible per se (starch, lipids), suggest that the cytotoxic effects of sample 3 and the magnetite cationic liposomes arise mainly from the electrostatic bindings between the particle coating and the negatively charged membrane components, such as N-acetyl-neuraminic acid residues, impairing normal cell functions, e.g. receptor-mediated signaling and endocytosis. On the other hand, it was demonstrated that DEAE-dextran led to high cytotoxicity during non-viral gene transfer experiments with epithelial cells [13], indicating that DEAE may also have had some influence on the survival of cells after incubation with sample 3. The increased cytotoxicity of sample 2 as compared to sample 3 is attributed to the influence of chemical components or non-physiologic pH. Cytotoxic effects were also found in cells when exposed to sample 1 having an anionic surfactant, even though the effects were lower as compared to samples 2 and 3. The cytotoxicity can be attributed to the anionic coating as well as to other components of the magnetic fluid. Being a proprietary of the vendor, the components of samples 1 and 3 were not known to us. Nevertheless, in vivo experiments with mice indicate that adsorption of carboxylic acids (tartrate anions) at the surface of manganese ferrite nanoparticles does not inexorably turn a magnetic fluid biocompatible, even at neutral pH and physiological salinity [14]. Furthermore, a decrease in the cell survival rates after an incubation period of 72 h at higher concentrations of the non-coated magnetite particles (sample 4, 20 ng/cell) was found. Since magnetite is known to be well tolerated by the
11
human body [10], the effects are attributed to an impairment of cellular functions due to a particle overload, even though no electrostatic bindings to cellular membranes occurred. The data indicate that subcellular damages were induced after a short incubation period (4 min) of cells with the analyzed magnetic particles, with the exception of the non-coated one. The morphologic findings are in agreement with the cell survival rates (20 ng magnetite/cell, Table 2) related to samples 2 and 3 showing lower values as compared to the non-coated particles (sample 4), but not by the cell survival rates values associated with sample 1. The reasons for the diverging results in connection with sample 1 were not known and must be cleared up in future studies. Taken together, our results show that the coated prototype magnetic particles, particularly those with a cationic surfactant, are cytotoxic to BT-20 cells. Therefore, a ranking for the cytotoxicity of the analyzed ferrofluid samples can be established: sample 2, 3, 1 and 4. Applied to the use of the magnetic particle samples 1–3 for magnetic thermoablation of tumors, our data indicate that not only heating-induced but also cytotoxic effects originated from the magnetic material itself could take place at the treatment site, where the magnetic particles will be accumulated. It is conceivable that the cytotoxicity of the aforementioned magnetic material will not be restricted to the tumor cells. Therefore, adverse systemic effects could take place, particularly if an extensive washout of particles from the target occurs. Therefore, samples 1–3 should not be adequate for treatments of tumors in humans. Nevertheless, the heating potentials of the analyzed samples were of peculiar interest. As the heating capabilities of a magnetite sample strongly depend on the particle size and microstructure determining its magnetic properties [8], new samples with particle features yielding a high SAR together with appropriate coatings should be elaborated.
Acknowledgements The authors wish to thank Dr. Robert Mu. ller for valuable discussions, Mr. Dipl.-Ing. Christian
12
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Bergemann (Chemicell) for providing a ferrofluidic sample, Mrs. Doreen Schro. der and Mrs. Uta Rother for excellent technical assistance. References [1] M. Boyer, L.E. Townsend, L.M. Vogel, et al., J. Vasc. Surg. 31 (2000) 181. [2] R. Bhatt, B. Scott, S. Whitney, R.N. Bryan, et al., Nucleosides Nucleotides 18 (1999) 1297. [3] J.P. Earls, D.A. Bluemke, MRI Clin. North Am. 7 (1999) 255. [4] A.S. Lu. bbe, C. Bergemann, H. Riess, et al., Cancer Res. 56 (1996) 4686. [5] R.K. Gilchrist, R. Medal, W.D. Shorey, et al., Ann. Surg. 146 (1957) 596.
[6] I. Hilger, W. Andr.a, R. B.ahring, et al., Invest. Radiol. 32 (1997) 705. [7] I. Hilger, R. Hergt, W.A. Kaiser, Invest. Radiol. 35 (2000) 170. [8] R. Hergt, W. Andr.a, C.G. d" Ambly, et al., IEEE Trans. Magn. 34 (1998) 3745. [9] I. Hilger, W. Andr.a, R. Hergt, et al., Radiology 218 (2001) 570. [10] G. Marchal, P. van Heke, P. Demaerel, et al., Am. J. Radiol. 152 (1988) 771. [11] T. Mosmann, J. Immunol. Methods 65 (1983) 55. [12] M. Shinkai, M. Yanase, H. Honda, et al., Jpn. J. Cancer Res. 87 (1996) 1179. [13] K. Abul-Hassan, R. Walmsley, M. Boulton, Curr. Eye Res. 20 (2000) 361. [14] Z.G.M. Lacava, R.B. Azevedo, L.M. Lacava, et al., J. Magn. Magn. Mater. 194 (1999) 90.
Cytotoxicity of selected magnetic fluids on human adenocarcinoma cells Ingrid Hilgera,*, Sylvia Fru. haufa, Werner Linb, Robert Hiergeistc, Wilfried Andr.ac, Rudolf Hergtc, Werner A. Kaisera a
Institut fu. r Diagnostische und Interventionelle Radiologie des Klinikums der Friederich-Schiller-Universita.t Jena, Bachstraße 18, D-07740 Jena, Germany b Institut fu.r Anatomie I der Friederich-Schiller-Universita.t Jena, Teichgraben 7, D-07740 Jena, Germany c Institut fu.r Physikalische Hochtechnologie Jena, Winzerlaer Straße 10, D-07740 Jena, Germany Received 3 January 2001; received in revised form 30 March 2001
Abstract Based on the knowledge that the magnetite particles seem to be well tolerated by the human body, the cytotoxic potential of coated particles was investigated, which had been selected for potential applications regarding the minimalinvasive elimination of breast tumors by magnetic thermoablation. Human adenocarcinoma cells (BT-20) were exposed (24, 48 and 72 h) to different magnetite particles with diverging total size (8, 10 and 220 nm) and coating (cationic and anionic). One sample contained only non-coated magnetite particles. The magnetite concentration ranged between 0.2 and 20 ng/cell. Cytotoxicity was estimated by measuring the succinate dehydrogenase activity. The morphologic features resulting from the interaction of magnetic fluids with BT-20 cells was determined by transmission electron microscopy. As opposed to the non-coated magnetic particles, cationic particles induced the strongest decrease in cell survival rates depending on time and concentration. Morphologically, the cationic particle samples exerted a strong binding to cellular membranes. Changes in the subcellular structure were found in relation to the coated magnetic particles. In conclusion, our results show that the coated prototype magnetic particles, particularly those with a cationic surfactant, are cytotoxic to BT-20 cells. The cytotoxicity is attributed to electrostatic bindings with cellular membranes, influences of chemical components or non-physiologic pH. Considering the in vivo applications, adverse systemic effects are conceivable and more biocompatible coatings for the selected magnetic particles should be elaborated. r 2001 Elsevier Science B.V. All rights reserved. Keywords: Magnetic fluids; Cytotoxicity; Magnetic thermoablation; Breast cancer; Cancer therapy; Heating
1. Introduction Since the past decade, there has been an increasing interest in the use of colloidal suspen*Corresponding author. Tel.: +49-3641-935037; fax: +493641-936767. E-mail address: [email protected] (I. Hilger).
sions of magnetic nanoparticles for quite different purposes, e.g. cell separation techniques [1], biochemical analysis [2], diagnostic [3] or therapeutic applications [4]. Considering the previous investigations (e.g. Ref. [5]), a procedure called magnetic thermoablation, is being elaborated that possibly allows the minimal-invasive elimination of breast tumors. The fundamentals of the method
0304-8853/03/$ - see front matter r 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 1 ) 0 0 2 5 8 - X
8
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Table 1 Characteristics of magnetic particles used in the present study. The specific absorption rate (SAR) is defined by the energy converted into heat by magnetic losses per time and magnetite mass and was measured for an alternating magnetic field of 400 kHz and 6.5 kA/m Sample
Average total particle size (nm)
SAR (W/g)
Components of coating
1 2 3 4
10 10 220 8
82 69 29 38
Organic components with negative surfactant Organic components with positive surfactant Starch derivative with positive (DEAE) surfactant None
are that after loading the tumor with a magnetic material (iron oxides, magnetite) and exposing the breast to an alternating magnetic field, heating is generated in order to kill the tumor cells. The basic feasibility of the technique has been studied in previous investigations [6,7]. An important prerequisite for applications in magnetic thermoablation is the heating capabilities of the magnetite samples to be used which were shown to vary considerably between each other [8]. The heating capabilities are described by the specific absorption rate (SAR) being the energy converted into heat by magnetic losses per time and mass. Using the selected prototype magnetic particles with adequate heating potentials, temperatures of up to 701C were recorded in adenocarcinomas implanted in mice [9]. Nevertheless, up to now it is not known if the prototype samples are biocompatible to living organisms. Whereas magnetite per se is known to be well tolerated by the human body (e.g. Ref. [10]), magnetite nanoparticles are mostly maintained in colloidal suspensions using particle coatings with largely unspecified biocompatibility features. Therefore, in the present investigation, we investigated the cytotoxic potency after incubation of human adenocarcinoma cells with different prototype colloidal solutions of magnetic nanoparticles which, according to previous investigations, are of interest for applications regarding magnetic thermoablation of breast tumors. 2. Materials and methods 2.1. Magnetic nanoparticle samples Four different water-based prototype magnetic nanoparticle suspensions (ferrofluids) were used
(samples 1, 2 and 4FFerrofluidics: Nu. rtingen, Germany; sample 3 was kindly provided by Chemicell, Berlin, Germany). The samples differed from each other by both the average total particle size and particle coating. The reference sample (sample 4) was composed of non-coated magnetite particles. The SAR of different magnetite samples was determined from time dependent calorimetric measurements in an alternating magnetic field (amplitude of 14 kA/m and 300 kHz frequency) [8]. The particle characteristics are summarized in Table 1. 2.2. Cell culturing An asynchronically growing human breast adenocarcinoma cell line (BT-20, Deutsches Krebsforschungszentrum: Heidelberg, Germany) was cultivated in an exponential monolayer growth. The cultures were maintained in MEM containing 10% (v/v) fetal calf serum (Life Technologies: Karlsruhe, Germany) and 1 mM sodium pyruvate (GibcoBRL: Karlsruhe, Germany). All cultures were routinely checked for mycoplasma contamination. Cells were used up to the 30th passage. 2.3. Determination of cell viability after exposure of BT-20 cells to magnetic nanoparticles Cells were seeded in 96 microtiter plates (density: 1 103 cells/well). After 24 h, the culture medium was replaced by the magnetic particles containing one. The magnetite concentration was adjusted to be 0.2, 2 and 20 ng/cell (corresponding to the concentrations between 2 and 200 mg/ml culture medium). After 24, 48 and 72 h of incubation, the cytotoxicity of the samples was
9
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
determined using the succinate dehydrogenase assay [11]. Survival rates (v) were calculated by the formula: v(%)=(t 100%) : k, where t means the absorbance (450 nm) of the exposed cells and k of the non-exposed control cells.
2.4. Determination of morphologic features after the exposure of BT-20 cells to magnetic nanoparticle samples 1 106 cells were seeded in culture flasks (25 cm2) and incubated under standard conditions. After 24 h, cells were washed 3 times with PBS, incubated with the different magnetic nanoparticle samples (magnetite concentration: 30 ng/cell, equivalent to 6 mg/ml culture medium) for 4 min under room temperature. After washing 4 times with PBS in order to remove unbound magnetic particles, cells were fixed with a 3% (v/v) glutaraldehyde (Agar Scientific LTD: Stansted/ Essex GB) solution and processed for electron microscopy examinations.
3. Results Different effects of the analyzed prototype ferrofluid samples on the cell survival rates were observed (Table 2). Sample 4 showed only a slight decrease of the cell survival rates at higher magnetite mass densities (e.g. 20 ng/cell). These effects were more pronounced with increasing incubation time. Similar relationships were observed for sample 1. Samples 2 and 3 having a cationic particle surfactant revealed both distinct time as well as magnetite-concentration dependent effects on the viability of cells. In particular, no viable cells were found after the application of 20 ng/cell of sample 2. Therefore, sample 2 seems to be more cytotoxic to cells than sample 4. Particularly, comparing the cell survival rates for a concentration of 20 ng/cell, the cell survival rates related to samples 2 and 3 were lower than those for samples 1 and 4. The electron microscopy examinations (Fig. 1) of cells incubated for 4 min at a concentration of 30 ng/cell showed a continuous congregation of
Table 2 Cell survival of BT-20 cells after incubation with different magnetite nanoparticle samples for 24, 48 and 72 h. Experiments were performed in triplicate Sample/magnetite concentration (ng/cell)
Cell survival rates after incubation times (in h) 24
48
72
Sample 1 0.2 2 20
9276 9674 8974
8573 8577 7974
8577 8878 7977
Sample 2 0.2 2 20
9775 375 070
8478 372 070
8875 070 070
Sample 3 0.2 2 20
10575 9374 6976
99711 9078 5975
9174 7674 3672
Sample 4 0.2 2 20
9974 9275 9275
10377 9577 9174
9775 9876 8277
10
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Fig. 1. Subcellular features after exposure of BT-20 cells to different magnetite nanoparticle samples. (1)–(4): results with regard to samples 1–4 (30 ng magnetite/cell for 4 min), respectively. In contrast to (4), cells depicted in (1)–(3) show altered cellular features. ArrowsFcontinuous concentration of mostly aggregated and electron-dense particles along the cell membranes; arrowheadsFirregularly distributed agglomerations of electron-dense particles at the plasmalemma; vFvacuoles; iFmembrane-delimited, electrondense bodies (presumably lysosomes); cFchromatin, ‘‘clotted’’F(2) and (3) or normalF(4); nFnucleoli, ‘‘clotted’’F(2) and (3), normalF(4); oFmitochondria, swollenF(3), normalF(4); eFvacuolar structures of the endoplasmic reticulum; dFdiscontinuous plasmalemma. Magnification: approximately 5000-fold (technically caused variations among 10%).
mostly clustered and electron-dense particles along the cell membranes exposed to cationic magnetic particles (samples 2 and 3) containing culture medium. In contrast to that, irregularly distributed agglomerations of electron-dense particles at the plasmalemma were found in cells exposed to (samples 4 and 1). The subcellular features after incubation with sample 1 revealed a homogeneous distribution of chromatin within the cell nuclei and vacuolar alterations of the endoplasmic reticulum together with rounded electron-dense inclusions in the cytoplasm. The mitochondria were found to be partially swollen. With regard to sample 2, subcellular alterations typical for dying cells were found, such as discontinuously distributed chromatin in the cell nuclei, predominating vacuoles in the cytoplasm and disrupted cell membranes. The subcellular alterations found in relation to sample 3 included the presence of mostly swollen mitochondria, widespread vacuolar structures, welldelimited vesicles and tubes of the endoplasmic
reticulum, membrane-delimited and electron-dense bodies (presumably lysosomes) in the cytoplasm. The chromatin was in parts homogeneously disseminated within the cell nuclei, and partially distributed like clods. With regard to sample 4, no cellular alterations were found: cell nuclei and cytoplasm were inconspicuous and the mitochondria intact.
4. Discussion The cytotoxic potency of the different prototype ferrofluids was assessed by the determination of the cell survival rates as well as by the morphologic features resulting from the interaction with cells. According to our data, the strongest cytotoxic potency, illustrated by time and magnetite-concentration dependent decreases of cell survival, was found for samples distinctively aggregating to cell membranes, as was observed with regard to the cationic magnetic material (samples 2 and 3).
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
The irregularly distributed particle accumulations in the proximity of cell membranes found in relation to samples 1 and 4 are mainly attributed to the preparation of artifacts during cell sample processing for electron microscopy examinations. According to Shinkai et al. [12], growth inhibition was not observed after incubation of a rat glioma cell line with 200 ng of magnetite cationic liposomes per cell, but at higher magnetite cell concentrations. The exposure time was up to 80 h. In comparison to that, our results indicate that samples 2 and 3 are of higher cytotoxicity than the aforementioned magnetite cationic liposomes. The components of the coating being biocompatible per se (starch, lipids), suggest that the cytotoxic effects of sample 3 and the magnetite cationic liposomes arise mainly from the electrostatic bindings between the particle coating and the negatively charged membrane components, such as N-acetyl-neuraminic acid residues, impairing normal cell functions, e.g. receptor-mediated signaling and endocytosis. On the other hand, it was demonstrated that DEAE-dextran led to high cytotoxicity during non-viral gene transfer experiments with epithelial cells [13], indicating that DEAE may also have had some influence on the survival of cells after incubation with sample 3. The increased cytotoxicity of sample 2 as compared to sample 3 is attributed to the influence of chemical components or non-physiologic pH. Cytotoxic effects were also found in cells when exposed to sample 1 having an anionic surfactant, even though the effects were lower as compared to samples 2 and 3. The cytotoxicity can be attributed to the anionic coating as well as to other components of the magnetic fluid. Being a proprietary of the vendor, the components of samples 1 and 3 were not known to us. Nevertheless, in vivo experiments with mice indicate that adsorption of carboxylic acids (tartrate anions) at the surface of manganese ferrite nanoparticles does not inexorably turn a magnetic fluid biocompatible, even at neutral pH and physiological salinity [14]. Furthermore, a decrease in the cell survival rates after an incubation period of 72 h at higher concentrations of the non-coated magnetite particles (sample 4, 20 ng/cell) was found. Since magnetite is known to be well tolerated by the
11
human body [10], the effects are attributed to an impairment of cellular functions due to a particle overload, even though no electrostatic bindings to cellular membranes occurred. The data indicate that subcellular damages were induced after a short incubation period (4 min) of cells with the analyzed magnetic particles, with the exception of the non-coated one. The morphologic findings are in agreement with the cell survival rates (20 ng magnetite/cell, Table 2) related to samples 2 and 3 showing lower values as compared to the non-coated particles (sample 4), but not by the cell survival rates values associated with sample 1. The reasons for the diverging results in connection with sample 1 were not known and must be cleared up in future studies. Taken together, our results show that the coated prototype magnetic particles, particularly those with a cationic surfactant, are cytotoxic to BT-20 cells. Therefore, a ranking for the cytotoxicity of the analyzed ferrofluid samples can be established: sample 2, 3, 1 and 4. Applied to the use of the magnetic particle samples 1–3 for magnetic thermoablation of tumors, our data indicate that not only heating-induced but also cytotoxic effects originated from the magnetic material itself could take place at the treatment site, where the magnetic particles will be accumulated. It is conceivable that the cytotoxicity of the aforementioned magnetic material will not be restricted to the tumor cells. Therefore, adverse systemic effects could take place, particularly if an extensive washout of particles from the target occurs. Therefore, samples 1–3 should not be adequate for treatments of tumors in humans. Nevertheless, the heating potentials of the analyzed samples were of peculiar interest. As the heating capabilities of a magnetite sample strongly depend on the particle size and microstructure determining its magnetic properties [8], new samples with particle features yielding a high SAR together with appropriate coatings should be elaborated.
Acknowledgements The authors wish to thank Dr. Robert Mu. ller for valuable discussions, Mr. Dipl.-Ing. Christian
12
I. Hilger et al. / Journal of Magnetism and Magnetic Materials 261 (2003) 7–12
Bergemann (Chemicell) for providing a ferrofluidic sample, Mrs. Doreen Schro. der and Mrs. Uta Rother for excellent technical assistance. References [1] M. Boyer, L.E. Townsend, L.M. Vogel, et al., J. Vasc. Surg. 31 (2000) 181. [2] R. Bhatt, B. Scott, S. Whitney, R.N. Bryan, et al., Nucleosides Nucleotides 18 (1999) 1297. [3] J.P. Earls, D.A. Bluemke, MRI Clin. North Am. 7 (1999) 255. [4] A.S. Lu. bbe, C. Bergemann, H. Riess, et al., Cancer Res. 56 (1996) 4686. [5] R.K. Gilchrist, R. Medal, W.D. Shorey, et al., Ann. Surg. 146 (1957) 596.
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