pubs.acs.org/Langmuir © 2010 American Chemical Society
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Structure and in Vitro Biological Testing of Water-Based Ferrofluids Stabilized by Monocarboxylic Acids Mikhail V. Avdeev,*,† Birte Mucha,‡ Katrin Lamszus,§ Ladislau Vekas, Vasil M. Garamus,‡ Artem V. Feoktystov,† Oana Marinica,^ Rodica Turcu,# and Regine Willumeit‡ †
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Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research (FLNP JINR), Dubna, Russia, ‡ GKSS Research Centre, Geesthacht, Germany, §University Medical Centre Hamburg Eppendorf (UKE), Germany, Center for Fundamental and Advanced Technical Research (CFATR), Romanian Academy, Timisoara Division, Timisoara, Romania, ^National Center for Engineering of Systems with Complex Fluids (NCESCF), University Politehnica Timisoara, Timisoara, Romania, and #National Institute R&D of Isotopic & Molecular Technologies (NIRDIMT), Cluj-Napoca, Romania Received November 26, 2009. Revised Manuscript Received January 18, 2010 Water-based ferrofluids (magnetic fluids) with double-layer steric stabilization by short monocarboxylic acids (lauric and myristic acids) are considered to be a potential source of magnetic nanoparticles in brain cancer (glioblastoma) treatment. Structure characterization in the absence of an external magnetic field is performed, including transmission electron microscopy, magnetization analysis, and small-angle neutron scattering with contrast variation. It is shown that despite the good stability of the systems a significant part of the magnetite nanoparticles are in aggregates, whose inner structure depends on the stabilizer used. In particular, an incomplete coating of magnetite particles is concluded in the case of myristic acid stabilization. The ferrofluids keep their structure unchanged when added to the cancer cell medium. The intracellular accumulations of magnetite from the ferrofluids added to cancer cell cultures as well as its cytotoxicity with respect to human brain cells are investigated.
1. Introduction Applications of magnetic nanoparticles for biomedical purposes were actively developed in the last decade.1-4 As an example, cancer treatment in what concerns controllable drug delivery,5,6 diagnostics (magnetic resonance imaging7), and therapy (magnetic hyperthermia8-11) can be mentioned. This raises the problem of synthesizing biocompatible ferrofluids or magnetic fluids (fine liquid suspensions of magnetic nanoparticles), which are stable and controllable in biological media under different conditions. The classical scheme of ferrofluid stabilization in nonpolar organic liquids12 is based on coating magnetite nanoparticles with a single adsorption layer of oleic acid (OA, C18H34O2), which belongs to a class of nonsaturated monocarboxylic acids. In polar (1) Mornet, S.; Vasseur, S.; Grasset, F.; Veverka, P.; Goglio, G.; Demourgues, A.; Portier, J.; Pollert, E.; Duguet, E. Prog. Solid State Chem. 2006, 34, 237–247. (2) Wuang, S. C.; Neoh, K. G.; Kang, E. -T.; Pack, D. W.; Leckband, D. E. Adv. Funct. Mater. 2006, 16, 1723–1730. (3) Duguet, E.; Vasseur, S.; Mornet, S.; Devoisselle, J. M. Nanomedicine 2006, 1, 157–168. (4) Proceedings of the 7th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, Vancouver, Canada, May 21-24, 2008; H€afeli, U., Zborowski, M., Eds.; J. Magn. Magn. Mater. 2009, Vol. 321. (5) Berry, C. C.; Curtis, A. S. G. J. Phys. Appl. Phys. 2003, 36, R198–R206. (6) Ito, A.; Shinkai, M.; Honda, H.; Kobayashi, T. J. Biosci. Bioeng. 2005, 100, 1–11. (7) Arbab, A. S.; Liu, W.; Frank, J. A. Expert Rev. Med. Dev. 2006, 3, 427–439. (8) Wust, P.; Gneveckow, U.; Johannsen, M.; B€ohmer, D.; Henkel, T.; Kahmann, F.; Sehouli, J.; Felix, R.; Ricke, J.; Jordan, A. Int. J. Hyperther. 2006, 22, 673–685. (9) Pradhan, P.; Giri, J.; Samanta, G.; Sarma, H. D.; Mishra, K. P.; Bellare, J. R.; Banerjee, R.; Bahadur, D. J. Biomed. Mater. Res., Part B 2007, 81, 12–22. (10) Fortin, J. -P.; Wilhelm, C.; Servais, J.; Menager, C.; Bacri, J. -C.; Gazeau, F. J. Am. Chem. Soc. 2007, 129, 2628–2635. (11) Brusentsov, N. A.; Nikitin, L. V.; Brusentsova, T. N.; Kuznetsov, A. A.; Bayburtskiy, F. S.; Shumakov, L. I.; Jurchenko, N. Y. J. Magn. Magn. Mater. 2002, 252, 378–380. (12) Rosensweig, R. E. Ferrohydrodynamics; Cambridge University Press: Cambridge, England, 1985
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liquids, one has to deal with the strong interaction of the carrier and the coating surfactants. This limits the use of the classical stabilization scheme and instead of a single surfactant layer requires additional (double) stabilization in an excess of the free second surfactant. In some cases (e.g., alcohols13-16), this results in very stable ferrofluids, where the sterical (noncharged) stabilization mechanism still prevails. In other cases such as water (the basis of biological media), the synthesis of systems with individual magnetic particles coated with a surfactant layer, which would provide steric stabilization, is still a problem and is reviewed in refs 16 and 17. Even in comparatively stable water-based ferrofluids with double-layer stabilization (volume fraction of magnetic material, jm, does not exceed several percent), complex aggregation on different scales was reported.14-20 Recently, some progress in the synthesis of concentrated waterbased magnetic fluids (jm > 10%) has been achieved21 for the double stabilization of nanomagnetite by saturated monocarboxylic acids with short carbon chains, such as lauric acid (LA, C12H24O2) and myristic acid (MA, C14H28O2). As shown,21 (13) Bica, D.; Vekas, L.; Avdeev, M. V.; Balasoiu, M.; Marinica, O.; Stoian, F. D.; Susan-Resiga, D.; T€or€ok, Gy.; Rosta, L. Prog. Colloid Polym. Sci. 2004, 125, 1–9. (14) Avdeev, M. V.; Aksenov, V. L.; Balasoiu, M.; Garamus, V. M.; Schreyer, A.; T€or€ok, Gy.; Rosta, L.; Bica, D.; Vekas, L. J. Colloid Interface Sci. 2006, 295, 100–107. (15) Vekas, L.; Bica, D.; Avdeev, M. V. China Particuol. 2007, 5, 43–49. (16) Vekas, L. Avdeev, M. V. Bica, D. Magnetic Nanofluids: Synthesis and Structure. In Nanoscience and Its Applications in Biomedicine; Shi, D., Ed.; SpringerVerlag and Tsinghua University Press: Berlin, 2009; pp 650-702. (17) Tombacz, E.; Bica, D.; Hajdu, A.; Illes, E.; Majzik, A.; Vekas, L. J. Phys. Cond. Mater. 2008, 20, 204103. (18) Shen, L.; Stachowiak, A.; Fateen, S.-E. K.; Laibinis, P. E.; Hatton, T. A. Langmuir 2001, 17, 288–299. (19) Wiedenmann, A.; Hoell, A.; Kammel, M. J. Magn. Magn. Mater. 2002, 252, 83–85. (20) Balasoiu, M.; Avdeev, M. V.; Aksenov, V. L.; Hasegan, D.; Garamus, V. M.; Schreyer, A.; Bica, D.; Vekas, L. J. Magn. Magn. Mater. 2006, 300, e225–e228. (21) Bica, D.; Vekas, L.; Avdeev, M. V.; Marinica, O.; Socoliuc, V.; Balasoiu, M.; Garamus, V. M. J. Magn. Magn. Mater. 2007, 311, 17–21.
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the obtained systems are not free of aggregation but nevertheless remain stable in the absence of an external magnetic field. The colloidal stability (pH effect and salt tolerance) of very dilute water-based ferrofluids with jm ≈ (0.2-2) 10-3% stabilized by monocarboxylic acids with special attention to physiological conditions was studied in ref 17. The combination of steric (formation of the second layer) and electrostatic (dissociation of OH groups) stabilization in these fluids was claimed to be dependent on the alkyl chain length of the surfactant used. This kind of information is quite important for doubly stabilized ferrofluids because strong dilution disturbs the required equilibrium between adsorbed and free surfactant molecules, thus affecting the fluid stability. In the present article, we study the concentrated samples with one of the highest jm values among those for available water-based ferrofluids with long-term stability (at least 1 year). The key point is that a significant increase in the stabilized fraction of magnetite when using LA and MA indicates that there should be specific structural peculiarities in these fluids as compared to other stabilization schemes. Quite good biocompatibility of short-chain monocarboxylic acids (in comparison, for example, with dodecylbenzenesulphonic (DBS) acid successfully used in stabilizing technical water-based ferrofluids21) opens up possibilities for probing the new class of water-based ferrofluids in biomedical applications. As an initial step, we consider the structural aspects of these ferrofluids with respect to their behavior in the concrete biological medium; brain cancer culture glioblastoma;which amounts to up to 30% of all brain tumors and is one of the most difficult malignancies to cure.22-24 First, a structural investigation of initial ferrofluids is performed, which includes transmission electron microscopy (TEM), magnetization analysis, and small-angle neutron scattering (SANS). Again, a special feature of the SANS experiments is the relatively high initial particle concentration of ferrofluid samples, which makes it possible to perform detailed contrast variation by diluting them with various mixtures of light and heavy water to one concentration (jm ≈ 1%), thus providing on the one hand full identity of the studied samples and on the other hand enough particle concentration for a sufficiently high signal/ background ratio. Then, the studied ferrofluids are added to the cancer cell culture to follow the penetration rate in the cancer cells by Berliner blue staining and atomic absorption spectroscopy (AAS). Also, questions of particular interest are the stability of magnetic particles in the cancer cell medium and their toxicity with respect to human brain cells.
2. Materials and Methods 2.1. Preparation of Initial Magnetic Fluids. Concentrated magnetic fluids are synthesized at the Laboratory of Magnetic Fluids of CFATR (Timisoara, Romania) according to the previously described procedure.21 The process of preparation consists of the coprecipitation reaction of solutions of FeSO4 and FeCl3 under atmospheric conditions, heating to 80 C with continuous stirring, the addition of NaOH (or a solution of NH4OH in approximately 35% excess), a surfactant coating of magnetite nanoparticles by the addition of LA or MA at 80 C, phase separation, decantation, washing of the resulting magnetic organosol (distilled water), the elimination of residual salts, the (22) Brockmann, M. A.; Ulbricht, U.; Gr€uner, K.; Fillbrandt, R.; Westphal, M.; Lamszus, K. Neurosurgery 2003, 52, 1391–1399. (23) Martens, T.; Schmidt, N. O.; Eckerich, C.; Fillbrandt, R.; Merchant, M.; Schwall, R.; Westphal, M.; Lamszus, K. Clin. Cancer Res. 2006, 12, 6144–6152. (24) Ulbricht, U.; Eckerich, C.; Fillbrandt, R.; Westphal, M.; Lamszus, K. J. Neurochem. 2006, 98, 1497–1506.
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dispersion of double-layer-coated magnetite particles in a weak solution of NaOH or NH4OH (pH correction), and the purification of the resulting magnetic fluid by magnetic decantation and filtration. The final samples are denoted as LA þ LA (jm = 11%) and MA þ MA (jm = 13%). 2.2. TEM and Magnetization Analysis. Transmission electron microscopy of highly dilute (1000 times) and dried ferrofluids is performed on the 1010 JEOL microscope (200 kV). Magnetization analysis is carried out on systems diluted to jm ≈ 1.5%. Magnetization curves are obtained using a VSM 880 vibrating sample magnetometer (DMS/ADE TechnologiesUSA; NCESCF) with a maximal applied field of 800 kA/m. The samples are put into plastic leak-tight cylindrical cells (5 mm height) placed on a special vibrating holder of the magnetometer. 2.3. SANS. The measurements are made on the SANS-1 instrument25 at steady-state reactor FRG-1 of the GKSS Research Centre (Geesthacht, Germany). A differential cross section per sample volume (scattering intensity) that is isotropic over the momentum transfer vector qB is obtained as a function of modulus q = (4π/λ)sin(θ/2), where λ is the neutron wavelength and θ is the scattering angle. Samples are put into 1-mm-thick quartz cells (Hellma) and are kept at 25 C during the experiments; no magnetic field is applied. Scattering curves are obtained for a fixed neutron wavelength of 0.83 nm (monochromatization Δλ/λ = 13%) and a series of sample-detector distances from 0.7 up to 9.7 m (detecting area 55 55 cm2, spatial resolution 0.7 0.7 cm2) over a q interval of 0.06-2.5 nm-1. The standard calibration procedure with a water sample is used. Contrast variation is carried out to study the inner structure of dispersed particles in the fluids in terms of the scattering length density (SLD) distribution. For this purpose, the initial samples are diluted 10-fold with different mixtures of light water (H2O) and heavy water (D2O) in a way in which the D2O content, η, in the final sample is varied from 0 to 90%. As background solutions in SANS measurements, the corresponding mixtures of H2O/D2O with the same D2O content are used. 2.4. Magnetite Absorption by Cancer Cells (Berliner Blue Staining and AAS). For all biological experiments, the ferrofluids are sterilized by filtration (ROTH, Rotilabo Spritzenfilter steril; pore size of 0.22 μm). The glioblastoma cells and astrocytes (provided by UKE) are grown in DMEM Glutamax-I medium (GIBCO) with 10 and 20% fetal bovine serum gold, respectively. The cells are incubated in a normal medium and in media with magnetite nanoparticles (after the addition of samples LA þ LA and MA þ MA at concentrations of 0.25 and 0.5 μL/mL to the normal medium) for 2 days at 37 C. For Berliner blue staining, the cells are washed three times with PBS buffer, fixed with 10% formaldehyde for 1 h, and then washed three times for 2 min with distilled aqueous potassium ferrocyanide (10% w/v); after 5 min, the same volume of hydrochloric acid (20%) is added. After 30 min, the cells are washed two times with distilled aqua and then dehumidified by an alcohol chain (60, 70, 80, and 96%, 2 min). Photographs are taken with a Nikon (Eclipse Ti) microscope at 20 magnification. For AAS, the cells are washed three times with PBS buffer and then trypsinized (4 mL). After 3 min, the enzyme reaction is stopped with the normal medium (6 mL). Then 50 μL is used for counting (two times). The main part of the cell suspension is centrifuged (1500 rpm, 5 min, 25 C). The cell pellet is diluted in bidistilled aqua to get a concentration of one million cells/mL. The probes are homogenized in the ultrasound water bath for (25) Zhao, J.; Meerwinck, W.; Niinkoski, T.; Rijllart, A.; Schmitt, M.; Willumeit, R.; Stuhrmann, H. Nucl. Instrum. Methods A 1995, 356, 133–137.
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Figure 1. TEM images of dried samples and corresponding histograms of the magnetite particle size distributions with fits (lines) and obtained parameters of eq 1.
45 min and are filtered through a circular filter for qualitative analysis (MN 615; Macherey-Nagel) and then measured two times in the AAS setup (Perkin-Elmer 3300; wavelength of 248.3 nm). 2.5. Cytotoxicity Tests. The cells are washed with PBS and then trypsinized (0.05% trypsin EDTA 1; GIBCO). After 3 min of incubation at 37 C, the enzyme reaction is stopped by adding the normal medium. The cell suspension is stirred and centrifuged (1500 rpm, 5 min, 25 C), and the cell pellet is resuspended in fresh normal medium. A number of cells (20 000) are taken in each well to start the cytotoxicity assay at day zero. The cells are incubated for 2 days at 37 C (relative humidity 95%, 21% O2 and 5% CO2) and then washed with PBS buffer and detached with 500 μL of trypsin (0.05% trypsin EDTA 1; GIBCO) by incubating at 37 C for 3 min. The reaction is stopped by adding normal cell culture medium (2 mL). The cell suspension is resuspended, and 50 μL of it is diluted in 10 mL of Coulter solution (Coulter Diluent II), which is used for cell counting. Each probe is analyzed two times by ANOVA with a significance level of 0.05, which is the Holm-Sidak method (multiple comparisons vs a control group).
3. Results and Discussion 3.1. Electron Microscopy. In TEM images (Figure 1), the magnetite cores of the particles in ferrofluids can be seen. The corresponding diameter histograms of individual magnetite particles obtained from the TEM images for these samples are described well in terms of the log-normal size distribution function: " # 1 ln2 ðd=d0 Þ ð1Þ Dn ðdÞ ¼ pffiffiffiffiffiffi exp 2S2 2πSd where parameters d0 and S are varied to get the best fits; the resulting values are given in Figure 1. One can see that the character of the TEM images is similar for the two samples whereas the most Langmuir 2010, 26(11), 8503–8509
probable size, d0, and polydispersity index, S, are slightly higher in the sample LA þ LA. 3.2. Magnetization Analysis. The magnetization curves show behavior close to that of superparamagnetic systems. In Figure 2, they are compared to the curves obtained previously21 for waterbased ferrofluids with DBS acid stabilization. However, it should be mentioned that the use of the polydisperse Langevin approximation with the log-normal particle size distribution26 over the whole range of the external magnetic field does not give good fits, as, for example, in the case of low-concentration nonpolar ferrofluids.27,28 This indicates that some of the magnetic particles are in aggregates, where magnetic moments interact sufficiently to disturb the Langevin behavior. Still, as a first approximation, the Langevin formalism with the use of the magnetization parameters is applied for the quantitative characterization of the magnetic size distribution function: 1=3 18χi kB T ð2aÞ dm ¼ μ0 πMd Ms 1=2 6kB T Ms d0 3 ¼ ð2bÞ μ0 πH0 Md 3χi H0 S ¼
1 3χi H0 1=2 ln 3 Ms
ð2cÞ
where kB is the Boltzmann constant, T is the absolute temperature, μ0 is the magnetic permeability of vacuum, Md = 4.46 105 A 3 m-1 is the saturation magnetization of magnetite, and Ms is (26) Rasa, M. Eur. Phys. J. E 2000, 2, 265–275. (27) Avdeev, M. V.; Bica, D.; Vekas, L.; Marinica, O.; Balasoiu, M.; Aksenov, V. L.; Rosta, L.; Garamus, V. M.; Schreyer, A. J. Magn. Magn. Mater. 2007, 311, 6–9. (28) Avdeev, M. V.; Bica, D.; Vekas, L.; Aksenov, V. L.; Feoktystov, A. V.; Marinica, O.; Rosta, L.; Garamus, V. M.; Willumeit, R. J. Colloid Interface Sci. 2009, 334, 37–41.
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Figure 2. Normalized magnetization curves of the studied samples are compared with the curves from ferrofluids stabilized by DBS acid. Table 1. Particle Size Characteristics of Ferrofluids as Revealed from Experimental Magnetization Curves in Figure 2 sample
Ms, kA 3 m-1
χi
LA þ LA MA þ MA DBS þ DBS LA þ DBS MA þ DBS
8.76 6.37 8.68 8.28 8.28
0.23 0.21 0.35 0.23 0.22
d*m, nm d0, nm 10.3 11.2 11.95 10.55 10.40
5.8 4.4 6.06 6.09 6.46
S 0.36 0.45 0.39 0.35 0.33
dm, nm σm, nm 6.2 4.9 6.54 6.48 6.81
2.3 2.3 1.84 2.34 1.58
the saturation magnetization of the system. Values of χi, H0, and Ms are experimentally obtained from the analysis of the asymptotic behavior of the curves: MðHÞjH f 0 ¼ χi H MðHÞjH f ¥ ¼ Ms ð1 -
ð3aÞ H0 Þ H
ð3bÞ
For polydisperse systems, the effective magnetic diameter d* m from the monodisperse Langevin approximation actually characterizes the mean diameter of the larger size fraction of the dispersed particles. Parameters d0 and S are related to the log-normal distribution (eq 1) and are obtained in the polydisperse Langevin approximation. The results are gathered in Table 1, where additionally the mean particle size, dm, and the mean square deviation, σm, are recalculated from experimental d0 and S for Dn(d) of type (eq 1). From the * , one can conclude that the fluids do comparison of χi and dm not principally differ in characteristic magnetic size. These parameters are larger than those in the case of very stable classical ferrofluids based on nonpolar organic carriers27,28 or polar carriers,13 which reflects some additional correlation in the orientation of magnetic moments interacting in the aggregates. The effect of aggregates in the polydisperse approximation is more pronounced. In comparison with the TEM data, it shifts d0 toward significantly lower values (an unrealistic one in the case of sample MA þ MA). The studied stabilizations result in lower values of χi and d*m as compared to a concentrated technical ferrofluid with DBS þ DBS stabilization (Figure 2 and Table 1) also showing13,21 nonLangevin behavior, which reveals a larger fraction of smaller particles (lower magnetic correlation) in the given case. It is interesting that these parameters for mixed combinations LA þ DBS and MA þ DBS (Figure 2 and Table 1) are close to those for stabilizations LA þ LA and MA þ MA. This shows that the first surfactant layer somewhat regulates the magnetic size distribution, especially concerning small particles. A similar conclusion 8506 DOI: 10.1021/la904471f
regarding the use of LA and MA was drawn for magnetite with a single-layer coating in nonpolar organic ferrofluids.27,28 3.3. SANS. The presence of aggregates in bulk ferrofluids is clearly observed in the SANS curves corresponding to different contents of D2O in the carrier (Figure 3). The SLDs of the surfactants (about 0.10 1010 cm-2) do not differ much from the SLD of water (-0.56 1010 cm-2), which means that in pure H2O the surfactant component in the fluid structures is almost matched and the scattering comes mainly from magnetite with an SLD of about 6.90 1010 cm-2. The addition of D2O with an SLD of 6.34 1010 cm-2 increases the contrast from surfactants against the carrier, so specific features appear in the scattering curves as compared to the H2O case. In accordance with the previous conclusions about strong aggregate effects, we fail in fitting the experimental curves to the model of separate core-shell particles imitating spherical magnetite cores (with the radius distributed according to eq 1) coated with a surfactant shell. Hence, the data in Figure 4 are treated in terms of the approach of modified basic functions,29 which was recently applied well30 to aqueous magnetic fluids with charge stabilization. The scattering intensity is considered in the following form F I~cs ðqÞ þ ðΔ~ F Þ2 I~c ðqÞ IðqÞ ¼ I~s ðqÞ þ Δ~
ð4Þ
Δ~ F ¼ F e -Fs
ð5Þ
where
is the modified contrast, the difference between the effective mean SLD of the particles, Fe, and the SLD of the liquid carrier, ~ c(q), I~s(q), and I~cs(q) comprise Fs. Modified basic functions I~ information about the nuclear and magnetic SLD distributions within particles as well as the polydispersity function.29 Our main interest here is I~c(q), which is the shape scattering function averaged over all particles in the fluid. It is worth mentioning that both components of the fluids (magnetite and surfactant) contribute to this function, in contrast to the scattering in pure H2O. In a standard way, effective match point Fe in modified contrast (eq 5) should be found from the minimum in the forward scattering intensity, I(0), as a function of the D2O content in the carrier. However, because of the aggregation effect neither the Guinier approximation nor the indirect Fourier transform can be reliably applied to find I(0) over the whole η interval (which mainly concerns η = 0.2-0.8 seen in Figure 3). As a first approximation, we use the dependence I(q) ≈ η at minimally measured q value. As shown in ref 29, the I~c(q) function, which is our main interest in this work, is invariant with respect to Fe. The parabolic minima found in I(q) ≈ η give effective match points η0 = 0.464 (corresponding SLD = 2.642 1010 cm-2) and η0 = 0.506 (corresponding SLD = 2.931 1010 cm-2) of D2O content for samples LA þ LA and MA þ MA, respectively. The modified basic functions are found from the experimental scattering curves minimizing the χ2-functional31 χ2 ¼
1 X ½Ik ðqÞ -I~s ðqÞ -ΔF~k I~cs ðqÞ -ðΔF~k Þ2 I~c ðqÞ2 N -3 k σk 2 ðqÞ
ð6Þ
where Ik(q) and σk(q) are the experimental scattering intensity and its error for the q value obtained for each kth modified contrast, (29) Avdeev, M. V. J. Appl. Crystallogr. 2007, 40, 56–70. (30) Avdeev, M. V.; Dubois, E.; Meriguet, G.; Wandersman, E.; Garamus, V. M.; Feoktystov, A. V.; Perzynski, R. J. Appl. Crystallogr. 2009, 42, 1009–1019. (31) Whitten, A. E.; Cai, S.; Trewhella, J. J. Appl. Crystallogr. 2008, 41, 222–226.
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Figure 3. SANS contrast variation for samples (a) MA þ MA and (b) LA þ LA. Sample labels correspond to the content of D2O in the carrier.
Figure 4. (a) Experimentally obtained I~c(q) basic functions for the two ferrofluids. The line shows IFT fits of the curves with parameters of Rg
= 15.1 ( 0.2 nm, I(0) = (5.8 ( 0.1) 10-20 cm3 (LA þ LA) and Rg = 10.7 ( 0.1 nm, I(0) = (2.1 ( 0.1) 10-20 cm3 (MA þ MA). (b) Pair distance distribution functions of particles found from I~c(q) basic functions (-) are compared with the pair distance distribution functions from curves with 0% D2O content (---) and from the TEM measurements of the individual particles ( 3 3 3 ).
respectively, and N is the total number of curves obtained for different contrasts. It should be mentioned that the obtained I~s(q) functions repeat the residual scattering curves well in the effective match point in accordance with eq 4, which proves the correctness of the minimization (eq 6). Resulting basic functions I~c(q) for the two ferrofluids are given in Figure 4, together with their indirect Fourier transform (IFT)32 in the form of the p(r) function. Because shape scattering function I~c(q) describes the effective homogeneous particles, the p(r) function is the pair distance distribution (PDD) averaged over the particle shapes. The maximal values, where the p(r) functions approach zero, correspond to the maximal aggregate sizes, which are 49 and 33 nm for samples LA þ LA and MA þ MA, respectively. The obtained values are in agreement with the dynamic light scattering data for similar samples17 with reported average hydrodynamic sizes of 77 and 48 nm for fluids LA þ LA and MA þ MA, respectively. In particular, the average size ratios between the two fluids are similar: 1.5 (SANS) and 1.6 (DLS). The radius of gyration, Rg, calculated from the p(r) functions (15.2 ( 0.2 nm (LA) and 10.2 ( 0.1 nm (MA)) is connected to the (32) Pedersen, J. S. Adv. Colloid Interface Sci. 1997, 70, 171–210.
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radius of gyration of the particle shape, Rc, and volume, Vc, as Rg2 = ÆRc2Vc2æ/ÆVc2æ, where the brackets denote the averaging over the particle radius distribution Dn(R). Assuming the quasispherical shape of the aggregates, which allows one to use the relation Rc2 = (3/5)R2, one obtains the characteristic radii of the particles (ÆR2Vc2æ/ÆVc2æ)1/2 to be equal to 19.5 ( 0.3 and 13.8 ( 0.1 nm for fluids LA þ LA and MA þ MA, respectively. For comparison, the PDD functions calculated from the TEM data of separate particles are also given in Figure 4b. Taking into account the mentioned difference in the I~c(q) function and the scattering intensity in the case of pure H2O (η = 0), we compare the corresponding PDD functions in Figure 4b. It is interesting that such a difference is clearly seen only in the LA þ LA sample. The maximal size from the I~c(q) function is shifted up to about 7 nm from that of the magnetite component, which can be related well to the effective thickness (about 3.5 nm) of the surfactant shell around magnetite nanoparticles. The obtained thickness exceeds the doubled length of the LA molecule, 1.4 nm, as calculated by the Tanford formula,33 which points to the bulky (nonoverlapped) structure of the (33) Tanford, C. J. Phys. Chem. 1972, 76, 3020–3024.
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Figure 5. Schematic view of the aggregate structure in the studied ferrofluids.
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Figure 6. Optical microscope images of cancer cell line G55 stained by Berliner blue after incubation (3 days) in a medium containing magnetic nanoparticles from sample MA þ MA (amount of added sample indicated at the top) as compared to the control cell culture.
stabilizing shell. In the MA þ MA sample, both kinds of PDD functions are very similar, so the surfactant shell does not have any effect. Such a possibility is schematically illustrated in Figure 5, which suggests a lower relative content of the surfactant component in the MA þ MA sample as compared to that in LA þ LA. The last fact is verified by the smaller value of the effective match point, which can be roughly associated with the mean particle SLD. Using the fact that SLDs of the surfactants are close to zero, one can estimate the volume fractions of magnetite, εm, from the match point to be F εm ≈ e ð7Þ Fm Here, Fe is assumed to be the mean aggregate SLD (corresponding to the effective match point), and Fm is the magnetite SLD. Such an estimate gives for εm values of 0.38 and 0.44 for samples LA þ LA and MA þ MA, respectively, showing less surfactant content (0.56 vs 0.62) in the second case. It is also seen that the effect of the surfactant shell in the SANS curves at the maximal D2O content in the carrier (Figure 3), which gives a specific band around q = 0.8 nm-1, is more pronounced in the LA þ LA sample. Therefore, one can conclude that the coating of magnetite particles in the aggregates of the MA þ MA sample is incomplete. Nevertheless, it slightly affects the stability of this fluid. A reasonable explanation is the presence of discrete (nonaggregated) particles in the solution (shown in Figure 5), which prevent the secondary association of the aggregates under discussion. The existence of separate particles in the studied fluids is confirmed by the comparatively high volume fraction of dispersed magnetite in the initial samples. It is also reflected in biological tests presented below in what concerns the incorporation of magnetite into cancer cells. The structure of both samples is fully preserved from the viewpoint of SANS when the initial systems are diluted with the cancer growth medium showing that the studied ferrofluids are stable in the biological medium. Also, the temperature increase (up to 80 C) does not reveal any significant change in the scattering, which indicates that the observed aggregates are quite stable formations. The important point is that the structure of the studied ferrofluids differs from that obtained after the first attempts of the described synthesis with the classical oleic acid.13,14 Ferrofluids with OA þ OA stabilization are characterized by larger (size >120 nm) aggregates with a developed fractal organization, which is reflected in a power-law behavior in the SANS curves.14 In this respect, they are close to technical water-based ferrofluids doubly stabilized by dodecylbenzenesulphonic acid.14,20,21 8508 DOI: 10.1021/la904471f
Figure 7. Absorption rate of magnetic particles by two lines of cancer cells (G55 and G112; UKE) obtained by ASS (3 days of incubation in a medium containing magnetic nanoparticles from two ferrofluids).
At the same time, aggregates in DBS-containing fluids are destroyed by increasing temperature14,15,34 but aggregates in stabilizations OA þ OA,14 LA þ LA, and MA þ MA (as noted above) are temperature-stable. This observation indicates that the binding of monocarboxylic acids in the stabilizing layer is quite strong as compared to their interaction with water molecules. It partially explains another significant difference between the currently studied systems and DBS-containing ferrofluids. DBS acid can be easily dissolved in water with formation of micelles.35 As a consequence, the required excess surfactant in the synthesis of ferrofluids results in the appearance of surfactant micelles in the final samples,20 thus reflecting a significant volume fraction (>1%) of nonadsorbed acid molecules. In contrast, no additional aggregates (micelles) of nonadsorbed surfactant are observed for (LA þ LA)- and (MA þ MA)-stabilized ferrofluids. 3.4. Biological Tests. The incorporation of magnetite particles into cancer cells as a result of endocytosis can be seen in optical microscope images of the samples stained with Berliner blue (Figure 6). The blue color is associated with iron bonded to (34) Balasoiu, M.; Avdeev, M. V.; Aksenov, V. L. Crystallogr. Rep. 2007, 52, 505–511. (35) Petrenko, V. I., Avdeev, M. V., Garamus, V. M., Bulavin, L. A., Aksenov, V. L. Rosta, L. Colloids Surf., A 2010, submitted for publication.
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4. Conclusions
Figure 8. Results of cytotoxicity tests: relative survival rate of different cancer lines and astrocytes after 3 days of incubation in media with ferrofluids with respect to the control (nonincubated) cultures.
the potassium ferrocyanide staining compound. The intensity of the absorbed magnetite correlates with the amount of ferrofluids initially added to the cancer medium. The normal iron content of cells (control) is below the limit of detection. Similar images are obtained for both ferrofluids in different cell lines. Quantitative analysis of the penetration rate of magnetite into the cells is given in Figure 7, where the typical ASS data for two arbitrary cell lines are shown. It is of the same order of magnitude for both particle sources, which proves that the number of individual (or slightly aggregated) particles is of the same order of magnitude in the initial ferrofluids but somewhat depends on the cell line. The cytotoxicity effect is presented in Figure 8. Together with the cancer cell culture, the astrocytes are also incubated under the same conditions. Again, the toxicity rate depends on the cancer cell line. In some cases, the incorporation of magnetic particles slows the culture growth to 50-60%, but in other cases, such incorporation is almost nontoxic. At the same time, on average one can conclude that the magnetic particles that are used have some selective toxic effect with respect to the cancer cells. This follows from the fact that the cytotoxicity of the studied particles for astrocytes is low and does not depend on the initial particle concentration. This correlates with the reported36,37 ability of astrocytes to incorporate and export iron actively. (36) Qian, Z. M.; Liao, Q. K.; To, Y.; Ke, Y.; Tsoi, Y. K. Cell. Mol. Biol. 2000, 46, 541–548. (37) Hoepken, H. H.; Korten, T.; Robinson, S. R.; Dringen, R. J. Neurochem. 2004, 88, 1194–1202.
Langmuir 2010, 26(11), 8503–8509
The structure of water-based ferrofluids with double-layer sterical stabilization by short monocarboxylic acids (lauric and myristic acids) is described relative to their possible biomedical applications, in particular, for cancer treatment. It is shown that besides discrete particles coated with a surfactant shell, a significant part of the particles are bound into aggregates, which are temperature-stable. The inner structure of the aggregates differs for the two ferrofluids with respect to the relative content of magnetite and surfactant, showing that in the case of myristic acid stabilization the magnetite particles are not fully coated with surfactant. Nevertheless, this factor does not have any significant effect on fluid stability in the absence of an external magnetic field. The obtained thickness of the surfactant shell in the case of lauric acid stabilization corresponds well to the double-chain length, thus confirming the existence of the double layer at the magnetite surface. Both fluids can be used well in cancer (glioblastoma) treatment. As a source of magnetic nanoparticles, they show high stability in the cancer cell medium and provide a high incorporation of magnetic nanoparticles into cancer cells, which is strongly dependent on the cancer cell line. Whereas the magnetic particles from the probed ferrofluids are characterized by very low toxicity in human brain cells, some increase in cytotoxicity can be seen on average with respect to cancer cells. We believe that the presented stabilization procedure of waterbased ferrofluids using short-chain-length monocarboxylic acids is a significant step in the development of magnetic carriers for biomedical applications. The main advantage of double-layer sterical stabilization is a comparatively low sensitivity to the chemical properties (pH and ionic strength) of the solutions.21 The considered ferrofluids are also characterized by a relatively low concentration of free (nonadsorbed) surfactant (as compared to other polar ferrofluids13,21), which is an important factor from the viewpoint of their biocompatibility. Acknowledgment. The studied magnetic fluids were synthesized by Dr. Doina Bica (1952-2008), who was one of the authors of the presented ideology. This work has been done in the framework of the HelmholtzRFBR project (HRJRG-016). This research has also been supported by the European Commission under the sixth Framework Program through Key Action: Strengthening the European Research Area, Research Infrastructures (contract no. RII3-CT2003-505925, GKSS, Germany) and by the Romanian Authority for Scientific Research through the Nanomagpoli research project. Special thanks are due to Dr. L. Barbu (Center of Electron Microscopy, University Babes-Bolyai Cluj-Napoca) for TEM images of ferrofluids.
DOI: 10.1021/la904471f
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